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: London South East RGC GSTT Approved: September Disease/condition approved name and Congenital myopathy subtypes: symbol as published on the OMIM Nemaline myopathy; NEM3, NEM5, NEM1, NEM4, database (alternative names will be listed on the NEM2, NEM6, NEM7 UKGTN website) Myopathy, centronuclear: CNM1, CNM2, CNMX Central core disease of muscle Myopathy, Congenital, with Fiber-Type Disproportion CFTD Arthrogryposis, distal; DA2B 2. OMIM number for disease/condition NEM3: , NEM5: , NEM1: , NEM4: , NEM2: , NEM6: , NEM7: , CNM1: , CNM2: , CNMX: , CCD: , DA2B: 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. The congenital myopathies are a group of inherited muscle diseases normally present at birth, marked by characteristic structural abnormalities in muscle fibres. The prognosis for affected individuals varies greatly according to the particular condition, ranging from the often neonatally lethal X-linked myotubular myopathy (XLMTM) to relatively mild cases with RYR1-related centronuclear myopathy. 4. Disease/condition mode of inheritance X linked/autosomal Dominant/Autosomal Recessive 5. Gene approved name(s) and symbol as published on HUGO database (alternative names will be listed on the UKGTN website) actin, alpha 1, skeletal muscle; ACTA1* bridging integrator 1; BIN1* cofilin 2 (muscle); CFL2* dynamin 2; DNM2* kelch repeat and BTB (POZ) domain containing protein 13; KBTBD13 myotubularin; MTM1* myosin, heavy chain 2, skeletal muscle, adult; MYH2 myosin, heavy chain 3, skeletal muscle, embryonic; MYH3 myosin, heavy chain 7, cardiac muscle, beta; MYH7 myosin, heavy chain 8, skeletal muscle, perinatal; MYH8 Nebulin; NEB ryanodine receptor 1 (skeletal); RYR1* selenoprotein N,1; SEPN1* troponin I type 2 (skeletal, fast); TNNI2 troponin T type 1 (skeletal, slow); TNNT1 troponin T type 3 (skeletal, fast); TNNT3 titin; TTN ORAI calcium release-activated calcium modulator 1; ORAI1

2 stromal interaction molecule 1; STIM1 stromal interaction molecule 2; STIM2 * = genes currently tested by Sanger sequencing 6. OMIM number for gene(s) ACTA1:102610, BIN1:601248, CFL2:601443, DNM2:602378, KBTBD13:613727, MTM1:300415, MYH2:160740, MYH3:160720, MYH7:160760, MYH8:160741, NEB:161650, RYR1:180901, SEPN1: TNNI2:191043, TNNT1:191041, TNNT3:600692, TPM2:190990, TPM3:191030, TTN:188840, ORAI1:610277, STIM1:605921, STIM2: Gene description(s) 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 N/A test being set up by Next Generation Sequencing; >1000 exons G (>100 amplicons; 40 MOLU) The test will detect point mutations in all coding exons including splice junctions. Whole exon deletions and duplications will be detected by read-depth analysis. Most mutations in most of the genes included are private. However, multiple founder effects have been documented for the SEPN1 gene (Ferreiro, Quijano- Roy et al. 2002) and some RYR1 mutations are recurrent, more common in some populations than others (Quane, Keating et al. 1994; Manning, Quane et al. 1998; Rueffert, Olthoff et al. 2001; Wilmshurst, Lillis et al. 2010). 9. Technical method(s) Next generation sequencing using Agilent SureSelect hybridisation capture of 22 genes of interest, followed by sequencing on Illumina platforms (HiSeq/GAIIx/MiSeq) The Agilent SureSelect in-solution array is used to capture by hybridisation all coding regions from all 22 genes, plus a minimum of 50 bp of intronic sequence either side of each exon. The captured DNA from each patient is prepared for sequencing which includes indexing using unique sequence tags for each sample so that the resulting sequence data can be identified as originating from a given sample. This enables efficiency savings through pooling of samples in the sequencing process. Sequence reads are separated by sample, and aligned to the human genome reference sequence or gene group region-of-interest references using NextGene software (SoftGenetics). Variants are flagged using NextGene software. SNPs present at high frequency in the general population (>=5%) are omitted from further analysis.

3 10. Validation process Please explain how this test has been validated for use in your laboratory The presence of all other identified variants (potentially pathogenic) detected in at least 10% of reads at a given locus are confirmed by fresh PCR amplification of individual exons and standard fluorescent capillary Sanger sequencing. Pathogenicity of confirmed variants is evaluated using the Alamut software application, as used for evaluation of variants detected by conventional capillary sequencing. Copy number variants (CNVs) are searched for by comparing the log2 ratio of relative read depth for each exon from each patient against the corresponding average value obtained from all samples in the same run. All target regions (coding exons + splice sites) will be covered by >30x read depth. Any targets that are covered by less than 30 reads will be subjected to standard PCR amplification and fluorescent capillary Sanger sequencing. Testing of relatives for identified familial mutations will be carried out by conventional PCR amplification and fluorescent capillary Sanger sequencing for point mutations, and by quantitative multiplex PCR assay for copy number variations (whole exon deletions or duplications), according to standard protocols used for other genes that we test in our laboratory. A number of the genes have been tested by Sanger/conventional sequencing in the laboratory for several years. The process of SureSelect enrichment followed by Illumina sequencing has been shown to be able to detect both point mutations and copy number changes using an early version of the capture design for these genes. The previous design has undergone full testing using positive controls for point mutations and large deletions; SNP and variant concordance was demonstrated at 20 positions. An updated design has since been shown to cover all targeted bases. The protocol to be used is identical to that applied to screening several genes causing glycogen storage disease, (Gene Dossier #65, approved ) 11a. Are you providing this test already? No but we have used Sanger sequencing for genes with asterisk in Gene section above. 11b. If yes, how many reports have you 1025 By Sanger sequencing produced? 11c. Number of reports mutation positive d. Number of reports mutation negative For how long have you been providing this service? 13a. Is there specialised local clinical/research expertise for this disease? 5 years Yes

4 13b. If yes, please provide details Prof. Francesco Muntoni Chair of paediatric neurology, Institute of Child Health, University College London Dr Heinz Jungbluth Senior Lecturer in Paediatric Neurology, Evelina Children s Hospital, Guy s & St. Thomas NHS Foundation Trust 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 150 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? 16a. Index cases b. Family members where mutation is known Based on experience how many tests will be required nationally (UK wide) per annum? Please identify the information on which this is based Yes the congenital muscular dystrophies and Duchenne Muscular dystrophy a. Index cases 200 (+/-100) 17b. Family members where mutation is 150 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. Approximately 200 (+/- 100) index cases. This figure is based on the existing throughput of around 200 index cases per year; although more genes are being offered, the current figure of ~200 includes cases resubmitted to the service for additional testing. We provide these tests as part of the NCG service, so expect that we already receive all national referrals for these genes. We are able to provide the National activity

5 EPIDEMIOLOGY 19. Estimated prevalence of condition in the general UK population Please identify the information on which this is based 20. Estimated gene frequency (Carrier frequency or allele frequency) 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. Based on small regional studies, the overall incidence of the congenital myopathies (including Central Core Disease, Multi-minicore Disease, Nemaline Myopathy and Centronuclear Myopathy) has been estimated at 0.06/1000 births (Orphanet J Rare Dis Sep 25;3:26) Allele frequency: (calculated from figure above) Penetrance figures are unavailable for most AD forms, however very variable expression is seen in RYR1- related myopathies and associated malignant hyperthermia susceptibility (Orphanet J Rare Dis Sep 25;3:26, Orphanet J Rare Dis. 2007, 2:25, Orphanet J Rare Dis. 2007, 2:31). Penetrance is variable in female carriers of MTM1 mutations (XL) and may depend on other genetic factors such as skewed X-inactivation (Orphanet J Rare Dis Sep 25;3:26) All patients tested will have a clinically, and in most cases histologically, diagnosed congenital myopathy, which would give a prevalence of close to 100% within this test population. There will be a small number of patients with an incorrect diagnosis of a congenital myopathy or with a different myopathy subtype. The mutation detection rate will be less than 100%; this capture includes all known genes for these myopathy subtypes, but it is known that there are as yet undiscovered genes contributing to this phenotype. INTENDED USE 23. Please tick the relevant clinical purpose of testing Diagnosis Yes No Treatment Yes No Prognosis & management Yes No Presymptomatic testing Yes No Carrier testing for family members Yes No Prenatal testing Yes 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. Please also see 9. for further details of analytical sensitivity and specificity. The analytical sensitivity is dependant on reliable capture of all target regions, and obtaining sufficient read depth at all target loci. We will follow quality control measures to ensure at least 30 reads are obtained per sample for each locus, to ensure reliable detection of heterozygous alleles. The analytical sensitivity to detect heterozygous changes at this read depth approaches 100% for single nucleotide variants and small rearrangements. In samples where clearly pathogenic mutation(s) are not identified, any loci which do not meet the required quality threshold for sequence reads will be PCR amplified and sequenced using conventional capillary electrophoresis sequencing. Potentially pathogenic variants detected in greater than or equal to 10% of reads will be confirmed by Sanger sequencing. Using this threshold, a relatively high proportion of false positives will be subjected to verification, however it is necessary to set the threshold at this level to eliminate the reasonable possibility of false negative calls; this figure can be revised once more data has been accumulated. 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). The remit of this test is to maximise the clinical sensitivity of testing by including all known genes for the conditions specified. Despite this, there are known to be associations to loci without a known causative gene (see references in section 21, above). Therefore sensitivity will be less than 100%. The clinical specificity may be less than 100% due to the identification of sequence variants of unknown significance. All attempts will be made to classify variants as accurately as possible, and this task will become easier as more data is generated, both locally and within published datasets, e.g. 1000genomes. 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). The positive predictive value should be very high (>99%) for the majority of the genes/conditions being tested, with the exception of variants identified in the RYR1 gene and a small proportion of female carriers of MTM1 mutations (see section 21, above). The negative predictive value will be high within a family with a known pathogenic mutation. 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 positive results for each part of the process. Please illustrate this with a flow diagram. The testing strategy is to simultaneously test all the genes by NGS. CLINICAL UTILITY 28. How will the test add to the management of the patient or alter clinical outcome? The test will help management and clinical outcome in subsets of patients; areas in which intervention can improve outcome include: management of ventilation support, monitoring of cardiac function in conditions with known heart involvement, anticipation of potential malignant hyperthermia susceptibility upon administration of anaesthetic and

7 choice of suitable pharmacological agents in selected conditions acetylcholinesterase inhibitors have been indicated as beneficial in centronuclear myopathy cases (Neuromuscul Disord Jun;21(6): Epub 2011 Mar 25) 29. How will the availability of this test impact on patient and family life? This test will help to provide a more timely diagnosis in a larger number of patients, since the previous testing repertoire consisted of a smaller subset of genes tested for in a sequential fashion. The increased number of molecular diagnoses will also help to provide family planning options in terms of prenatal and potentially preimplantation genetic diagnosis. 30. Benefits of the test Please provide a summary of the overall benefits of this test. The main benefits of this test are to provide an overall higher mutation detection rate in a faster time for patients with a congenital myopathy, compared with the current service. The higher detection rate will be as a result of testing a more comprehensive repertoire of genes, as well as the potential for the detection of large rearrangements, which is currently unavailable for the majority of these genes. The faster turn-around time is based on the simultaneous analysis of the gene set, as opposed to the previous strategy of sequential analysis. In addition, this test is predicted to provide an overall reduction in costs per patient, which will help to provide additional resources to other areas of the service. The cost of providing this service is predicted to decrease with time as the next generation sequencing reagents become cheaper. 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. The testing of the congenital myopathies is currently conducted as part of a multi-disciplinary service encompassing clinical, immunohistochemical/histological and molecular analysis. The pathological element to the service is typically carried out on a muscle biopsy, which can lead to a smaller number of potentially implicated genes. However the process of muscle biopsy is invasive and can have a detrimental outcome where the patients are particularly unwell and/or sensitive to anaesthetic or trauma. This test may therefore be useful for the shifting of emphasis or order of these processes so that these invasive procedures may be unnecessary for a subset of these patients. 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 genes. Patients should be appropriately consented within the genetic counselling clinic and unexpected results should be discussed with them. 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 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. The costing evaluation for this test is very difficult to calculate. We already test for a number of the genes included in this gene panel, so this represents a change in methodology for those tests, and cost savings will be based primarily on the difference in cost between methodologies, rather than additional savings from other clinical procedures. However, for the new genes within the panel which are currently not available as NHS tests, we do not know what the % mutation detection rate will be among our referrals. This test is predicted to increase the overall mutation detection rate, simply by testing more candidate genes for every referral and being able to detect a wider range of mutation types. Also the different referral types which are likely to have mutations in these new genes will (normally) undergo a variety of different clinical procedures prior to genetic testing, and until we know the relative proportions

8 of mutations being detected by gene and by clinical sub-type it is not possible to calculate overall savings. The cost of next generation sequencing is constantly decreasing. The price shown for this test of 1000 is based on current costings, however we anticipate being able to reduce this to between later this year. 35. List the diagnostic tests/procedures that would no longer be required with costs. Costs and type of imaging procedures Costs and types of laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Costs and types of physiological tests (e.g. ECG) Cost and types of other investigations/procedures (e.g. biopsy) Total cost tests/procedures no longer required 36. REAL LIFE CASE STUDY In collaboration with the clinical lead, describe a real case example to illustrate how the test would improve patient experience A patient with Multi-minicore Disease (MmD), a congenital myopathy to date mainly associated with mutations in the SEPN1 and RYR1 genes, was sequenced on a previous version of the capture being submitted in this dossier. Prior to capture and sequencing, the patient had undergone sequence analysis of the RYR1, SEPN1, ACTA1 and LMNA genes, as well as investigation of the 4q repeat region associated with facioscapulohumeral dystrophy (FSHD). The results indicated a putative mutation in the MYH7 gene. Further investigation of the family history showed a high frequency of early death, presumed to be of cardiac origin. Mutations in MYH7 are known to cause both hypertrophic cardiomyopathy and, less frequently, distal myopathies. The identification of a mutation in MYH7 is therefore in keeping with the myopathy as well as the cardiac involvement in this patient. This is however the first instance of an MYH7 mutation being associated with the typical histopathological appearance of MmD (manuscript submitted for publication). This case highlights the scope for uncovering new genotype-phenotype associations by the simultaneous analysis of genes involved in conditions with overlapping phenotype. This patient underwent numerous investigations over many years before this molecular diagnosis was made. The initial application of sequence capture and NGS could have provided a diagnosis for this patient much sooner than sequential analysis of candidate genes. In this case, this would have had the advantage of allowing surveillance for potential cardiac complications for the patient and for other family members. 37. For the case example, if there are cost savings, please provide these below: The costs for the individual molecular tests (RYR1, SEPN1, ACTA1, LMNA, FSHD) could have been avoided if the next generation sequencing approach had been available. Under current costs, the total cost of testing these 5 genes amounted to Combined with the cost of NGS testing ( 1000) a total of 3476 was spent in defining the molecular cause of this patient s myopathy. This could have cost only 1000 by applying the NGS screen, thereby saving 1476 on genetic tests alone. The patient also would have been subjected to fewer appointments and tests associated with the cardiac complications.

9 PRE GENETIC TEST Costs and type of imaging procedures Costs and type of laboratory pathology tests (other than molecular/cyto genetic 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 2476 POST GENETIC TEST Costs and type of imaging procedures Costs and types laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Cost of genetic test proposing in this gene dossier 1000 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 gene tests (RYR1,SEPN1, ACTA1, LMNA, FSHD) 38. Estimated savings for case example described 1476 (on genetic tests alone)

10 UKGTN Testing Criteria Approved name and symbol of disease/condition(s): See Table 1 Approved name and symbol of gene(s): See Table 2 OMIM number(s): See Table 1 OMIM number(s): See Table 2 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 Paediatric Neurologist Consultant Neurologist Consultant Clinical Geneticist Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Clinical features of a congenital myopathy OR Tick if this patient meets criteria Histopathological features of a congenital myopathy on muscle biopsy 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

11 Table 1 Approved name and symbol of disease/condition(s): OMIM number(s): Congenital myopathy subtypes: Nemaline Myopathy 3; NEM Nemaline Myopathy 5; NEM Nemaline Myopathy 1; NEM Nemaline Myopathy 4; NEM Nemaline Myopathy 2; NEM Nemaline Myopathy 6; NEM Nemaline Myopathy 7; NEM Myopathy, centronuclear: Myopathy, Centronuclear, 1; CNM Myopathy, Centronuclear, 2; CNM Myopathy, Centronuclear, X-Linked; CNMX Central Core Disease of Muscle Myopathy, Congenital, With Fiber-Type Disproportion; CFTD Arthrogryposis, Distal, Type 2b; DA2B Table 2 Approved name and symbol of gene(s): OMIM number(s): actin, alpha 1, skeletal muscle; ACTA1* bridging integrator 1; BIN1* cofilin 2 (muscle); CFL2* dynamin 2; DNM2* kelch repeat and BTB (POZ) domain containing protein 13; KBTBD13 myotubularin; MTM1* myosin, heavy chain 2, skeletal muscle, adult; MYH myosin, heavy chain 3, skeletal muscle, embryonic; MYH myosin, heavy chain 7, cardiac muscle, beta; MYH myosin, heavy chain 8, skeletal muscle, perinatal; MYH Nebulin; NEB ryanodine receptor 1 (skeletal); RYR1* selenoprotein N,1; SEPN1* troponin I type 2 (skeletal, fast); TNNI troponin T type 1 (skeletal, slow); TNNT troponin T type 3 (skeletal, fast); TNNT tropomyosin 2 (beta); TPM2* tropomyosin 3; TPM3* titin; TTN ORAI calcium release-activated calcium modulator 1; ORAI stromal interaction molecule 1; STIM stromal interaction molecule 2; STIM * = genes currently tested by Sanger sequencing