Proposal form for the evaluation of a genetic test for NHS Service Gene Dossier/Additional Provider TEST DISORDER/CONDITION POPULATION TRIAD Submitting laboratory: Leeds RGC Approved: Sept 2013 1. Disorder/condition approved name and symbol as published on the OMIM Malignant Hyperthermia, susceptibility to, 1 (MHS1) Malignant Hyperthermia, susceptibility to, 5 (MHS5) database (alternative names will be listed on the (Note, malignant hyperthermia is also known as UKGTN website) malignant hyperpyrexia) 2. OMIM number for disorder/condition 145600, 601887 3a. Disorder/condition please provide, in laymen s terms, a brief (2-5 sentences) description of how the disorder(s) affect individuals and prognosis. 3b Disorder/condition if required please expand on the description of the disorder provided in answer to Q3a. MH susceptibility predisposes to a potentially fatal reaction to the most commonly used anaesthetic drugs. Susceptible individuals are usually healthy but exposure to the triggering drugs causes a loss of calcium regulation in skeletal muscle cells leading to muscle spasm, a profound increase in metabolic activity and muscle cell disruption. Survival depends on prompt recognition of the evolving reaction and appropriate treatment by the anaesthetist. If a patient is known to be at increased risk for MH, a reaction can be avoided by the use of alternative anaesthetic drugs that are known not to trigger the condition. While most patients susceptible to MH are apparently healthy unless exposed to the triggering anaesthetics, there are some MH susceptible individuals who present in other ways. Known associations with MH susceptibility are exertional rhabdomyolysis, hyperckaemia and exertional heat illness. Patients with congenital myopathies with a RYR1 aetiology (central core disease, multiminicore myopathy) are also at risk of developing malignant hyperthermia if their RYR1 mutation results in gain of function of the RyR1 channel. Loss of function RYR1 mutations are not associated with MH susceptibility. There are also rare reports of MH susceptible children with nonspecific clinical and histological myopathic features who present with apparently spontaneous episodes of fever and muscle rigidity that can prove fatal. Finally late onset myopathy has also recently been described as a presenting feature of MH susceptibility. 4. Disorder/condition mode of inheritance MH susceptibility was originally described as an autosomal dominant condition with incomplete penetrance. There is mounting evidence that MH susceptibility is one of a growing number of presumed single gene disorders that deviates from a simple Mendelian model of inheritance. Indeed, inheritance of MH can be best explained by a threshold model: approximately 8% of UK MH families appear to have more than one genetic factor making a major contribution to the risk of MH susceptibility.
5. Gene approved name(s) and symbol as published on HGNC database (alternative names will be listed on the UKGTN website) ryanodine receptor 1 (skeletal); RYR1 calcium channel, voltage-dependent, L type, alpha 1S subunit; CACNA1S 6a. OMIM number for gene(s) 180901, 114208 6b HGNC number for gene(s) 10483, 1397 7a. Gene description(s) RYR1, chromosomal location 19q13.1, encodes the skeletal muscle ryanodine receptor isoform. This protein forms the monomeric subunits of the tetrameric calcium release channel of skeletal muscle sarcoplasmic reticulum. The gene encompasses 158 Kb gdna and contains 106 exons with the cdna (~15 Kb) encoding a protein monomer of 5038 amino acids with a mass of 563,584 Da. CACNA1S, chromosomal location 1q32, encodes the pore-forming subunit of the pentameric calcium channel/voltage sensor of the skeletal muscle sarcoplasmic membrane. The gene encompasses 73 Kb gdna and contains 44 exons with the cdna (~ 5.6 Kb) encoding a protein of 1873 amino acids with a mass of 212,350 Da. 7b. Number of amplicons to provide this test (molecular) or type of test (cytogenetic) 7c. GenU band that this test is assigned to for index case testing 8. Mutational spectrum for which you test including details of known common mutations 32 long PCR fragments (23 for RYR1 and 9 for CACNA1S) to include all exons and intronic boundaries. G (50-100 amplicons) Analysis of entire coding region and splice junctions for all potentially deleterious variants (which we define as missense, nonsense, indels, or in a putative splice site) in RYR1 and CACNA1S. Of these, 30 RYR1 (www.emhg.org) and 2 CACNA1S (Monnier et al. Am J Hum Genet 1997; 60: 1316-25; Weiss RG et al Am J Physiol Cell Physiol 2004; 287: C1094-102; Carpenter D et al BMC Med Genet 2009; 10:104; Eltit JM et al Proc Nat Acad Sci 2012; 109: 7923 8) have been functionally characterised and meet international consensus criteria for diagnostic use in MH. 9a. Technical method(s) Panel test using NGS involving clonal sequencing of indexed multiplexed samples. PCR amplification of coding and flanking (minimum 20bp) intronic sequence. NGS analysis using Illumina (HiSeq or MiSeq) platform. 9b If a panel test using NGS please state if it is a conventional panel or a targeted exome test. 9c. Panel/targeted exome Tests i) Do the genes have 100% coverage? If not what is the strategy for dealing with the gaps in coverage? Gene-centric approach for RYR1 and CACNA1S We have sequenced RYR1 and CACNA1S in 48 samples. For RYR1 there is 96-99% coverage, with 95-100% coverage of CACNA1S. There is an average read depth of 200. Missing fragments are sequenced using Sanger sequencing.
ii) Does the test include MLPA? iii) Does this use sanger sequencing or Next Generation Sequencing (NGS)? iv) If NGS is used, does the lab adhere to the Practice Guidelines for NGS? 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. 11. Validation process Please explain how this test has been validated for use in your laboratory or submit your internal validation documentation No NGS Yes Provided by the lab 12a. Are you providing this test already? No x Yes 12b. If yes, how many reports have you produced? Please provide the time period in which these reports have been produced and whether in a research or a full clinical diagnostic setting. Next generation sequencing workflow has been validated and applied to provision of our BRCA and HNPCC cancer services. The approach was been validated in a pilot study of 55 cases (Morgan et al., 2010 Human Mutation 31:484-491). An additional study of 50 cases analysed in parallel with Sanger sequencing was fully concordant for 485 variants (100% sensitivity). See details below of statistical analysis (section on analytical sensitivity and specificity). All long range primers were checked for SNPs. A minimum sequence coverage threshold was set at depth of 50 sequence reads. Diagnostic Next Generation sequencing pathways for BRCA and HNPCC genes have been established in service for 2.5 years and 1.5 years respectively. This workflow was also audited by the peer assessors during our most recent laboratory CPA assessment (May 2010). Have provided a diagnostic service for functionally characterised RYR1 mutations (www.emhg.org) in a full clinical diagnostic setting since 2003 (1020 samples tested). In a research setting we have carried out Sanger sequencing of cdna extracted from skeletal muscle of RYR1 (192 samples) and CACNA1S (50 samples) since 2004 and 2007 respectively. We commenced NGS sequencing of RYR1 and CACNA1S in a research setting in April 2012 (48 samples) 12c. Number of reports mutation positive 470 (468/1260 RYR1, 2/98 CACNA1S) 12d. Number of reports mutation negative 790 13. For how long have you been providing See 12b above this service? 14a. Is there specialised local clinical/research expertise for this disorder? No x Yes 14b. If yes, please provide details The Leeds MH Investigation Unit has been the national referral centre since 1971. With more than
15. Are you testing for other genes/disorders/conditions closely allied to this one? Please give details 6000 patients tested by muscle biopsy and/or DNA analysis, the Leeds unit has the greatest experience of MH diagnosis worldwide: it remains the most active service internationally with more than 150 patients (possible index cases and relatives of known cases) tested per year. Professor Hopkins has worked in the unit since 1988 and been its director since 2001. Further details of the service are attached. Since its foundation the Leeds MH Unit has combined research with its clinical service and this research has had a molecular genetic focus since 1990. Over the past 10 years research in the MH Unit has been funded by the Department of Health Pharmacogenetics programme, The Big Lottery Fund, the MRC and Wellcome Trust. Since 2012 the Leeds MH Unit has been a Core Component (PI Hopkins) of a US NIH Program Project Grant. See service description 16. Based on experience what will be the national (UK wide) activity, per annum, for: 16a. Index cases 75 16b. Family members where mutation is 100 known 17a. Does the laboratory have capacity to provide the expected national activity? Yes use of NGS for mutation detection will make the service already provided more efficient in terms of staff resources. 17b. If your laboratory does not have capacity 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. 18. Please justify the requirement for another laboratory to provide this test e.g. insufficient national capacity. N/A
EPIDEMIOLOGY 19a. Estimated prevalence of condition in the general UK population 19b. Estimated incidence 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 population of people that meet the Testing Criteria. Based on the incidence of MH in children (the age group with highest incidence), the likelihood of a child with MH susceptibility reacting during general anaesthesia and the number of children receiving general anaesthesia per year, we estimated the prevalence in the UK to be 1 in 8,000 10,000. The number of new cases of malignant hyperthermia presenting with a clinical reaction has averaged approximately 25 per year over the last 10 years. This represents an incidence of 1 in 2.4 million in the general population. However, only approximately 3 million people receive a general anaesthetic each year and so the incidence in the anaesthetic population is 1 in 120,000. The age distribution of those developing a reaction is skewed towards children and young adults, with an incidence of MH in children having general anaesthesia of 1 in 20,000 50,000. These data are based on referral patterns to the Leeds MH Unit (national referral centre) and are similar to epidemiological data from other countries. Using genetic data, Monnier and colleagues (ref) in France estimated the gene frequency at 1 in 3,000. Using similar methodology, we calculated a similar gene frequency for the UK. By definition MH can only occur in a patient receiving general anaesthesia and then only if the triggering drugs are used. Many patients react on their first anaesthetic exposure but some have had multiple general anaesthetics without apparent problems before subsequently reacting. The MH susceptibility phenotype, as defined by the in vitro contracture tests, is completely penetrant. For index cases who have been phenotyped MH susceptible using the in vitro contracture test, the prevalence is 100%. For index cases not tested initially by muscle biopsy, the prevalence will depend on which of the testing criteria they fulfilled. For example, in patients who developed a well documented fulminant metabolic reaction with rhabdomyolysis the prevalence is >95%. For those who present with a history of unexplained postoperative pyrexia the prevalence is approximately 1%. The overall prevalence for patients meeting any of the testing criteria is 30%.
INTENDED USE 23. 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. Diagnosis x Yes No Treatment x Yes No Prognosis & management x 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
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. Data 1) Total number of known variants by Gold standard (Sanger sequencing) re-tested by clonal sequencing: 485 Total number confirmed by clonal sequencing: 485 2) Number of unique variants (i.e. each variant counted once only) by Gold standard (Sanger sequencing) re-tested by clonal sequencing: 78 Total number confirmed by clonal sequencing: 78 Analysis Determined by binomial confidence interval method (see http://statpages.org/confint.html). Using 95% confidence interval (2.5% in each tail). 1) Total number of variants (n=485) As no variants have been missed, binomial distribution predicts we can be 95% confident that the false negative rate is below 0.76%. However, it may be unrealistic to assume all variants are equally detectable, so considering each variant once only is a more cautious approach. 2. Number of unique variants The most cautious approach to validating clonal sequencing sensitivity would be to test a different variant each time, since re-testing a variant which is already know to be detectable is of limited value. Therefore, a repeat analysis was carried out counting only the total number of unique variants (n=78). Again, as no variants have been missed, binomial distribution predicts we can be 95% confident that the false negative rate is below 4.62%. Standard assumptions apply including: Quality standards for validation work are the same for subsequent patient tests. Testing criteria and methods remain unchanged or modifications do not have an impact on sensitivity. Samples tested (or the range of variants tested in validation) are representative of patients (or variants) that will be tested. Specificity of Illumina sequencing Common artefacts have been observed on numerous occasions at single nucleotide runs (As and Ts). Where these are detected at a significant level Sanger sequencing is used to exclude the presence of a variants. 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 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). For characterised diagnostic mutations (www.emhg.org): Sensitivity: 40% Specificity: 95%
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). For those with previous phenotyping using the in vitro contracture test: PPV, for those with MH susceptible phenotype= 100% NPV, for those with MH negative phenotype = 100% For those meeting the Test criteria with DNA analysis as the initial test PPV = 77% NPV = 79% 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. This will be added to the published Testing Criteria. Deep resequencing of RYR1 and CACNA1S will be done simultaneously. CLINICAL UTILITY 28. How will the test change the management of the patient and/or alter clinical outcome? A positive test will avoid the need for an open muscle biopsy, which involves a 2 night stay in Leeds. The biopsy is an open surgical procedure that leaves a scar of 5 8 cm and discomfort for up to two weeks. The average period of absence from work for the biopsy is 7 days. 29. Benefits of the test for the patient & other family members Please provide a summary of the overall benefits of this test. The test provides diagnosis without the need for open muscle biopsy. Diagnosis is important because it enables the appropriate choice of anaesthetic drugs for safe anaesthesia while minimising side-effects and complications. 30. What will be the consequences for patients and family members if this test is not approved? The only means of diagnosis will be the muscle biopsy procedure in Leeds 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. See above 32. Please describe any specific ethical, legal or social issues with this particular test. MH susceptibility is generally a bar to recruitment to the military. There has always been a concern about the provision of safe anaesthesia for MH susceptible recruits on deployment. The more recent evidence that MH susceptible individuals are at increased risk of exertional rhabdomyolysis and possibly exertional heat illness has consolidated the situation. Serving members of the armed services who are newly diagnosed with MH susceptibility are handled on an individual basis in respect of their military careers. There may be other careers involving exposure to heat stress where risk to MH susceptible individuals is greater than normal.
33. Only complete this question if there is previously approved Testing Criteria and you do not agree with it. Please provide revised Testing Criteria on the Testing Criteria form and explain here the changes and the reasons for the changes. 34. List the diagnostic tests/procedures that an index case no longer needs if this genetic test is available. Type of test Cost ( ) Costs and type of imaging procedures Costs and types of laboratory pathology tests (other than molecular/cyto genetic test proposed in this gene dossier) Costs and types of physiological tests (e.g. ECG) Cost and types of other investigations/procedures (e.g. biopsy) Open muscle 3,500 biopsy with in vitro muscle contracture tests on the excised specimens and histopathological examination Total cost tests/procedures no longer required 3,500 (saving for people who are positive from genetic test) 35. Based on the expected annual activity of index cases (Q15a), please calculate the estimated annual savings/investments based on information provided in Q33. Number of index cases expected annually 75 Cost to provide tests for index cases if the genetic test in this gene dossier was not available (see Q34) 3,500 Total annual costs pre genetic test 262,500 Total annual costs to provide genetic test 39,750 Total savings 45 will still require the other tests 45x 3500 = 157,500 Total costs 39750+157500= 197,250 262500-197250= 65,250 saving 36. REAL LIFE CASE STUDY In collaboration with the clinical lead, describe TWO real case examples: 1. prior to availability of genetic test 2. post availability of genetic test to illustrate how the test improves patient experience and the costs involved. Case example one pre genetic test Patient x was 4 years of age when he sustained a supracondylar fracture of his arm requiring urgent manipulation under general anaesthesia. As part of the anaesthetic procedure he was given suxamethonium which resulted in difficulty in opening x s mouth rather than causing the expected muscle relaxation. The jaw muscle relaxed after about two minutes and the operation continued with anaesthesia maintained using isoflurane. Towards the end of the 20 minute operation the anaesthetist became concerned that x s heart rate and end-tidal CO2 levels were increasing. However, as the surgery soon finished, isoflurane was discontinued and was woken up. The heart rate settled and other observations in the recovery period were normal other than a temperature of 37.8 C.
As x was too young to undergo the muscle biopsy procedure, both parents were referred for MH testing. The father tested negative and the mother positive. PRE GENETIC TEST COSTS Costs and type of imaging procedures Costs and type of laboratory pathology tests Costs and type of physiological tests (e.g. ECG) Cost and type of other investigations/procedures (e.g. biopsy) Type of test 2 x Open muscle biopsy with in vitro muscle contracture tests on the excised specimens histopathological examination and Cost 7,000 Cost outpatient consultations (genetics and non genetics) Total cost pre genetic test 7,000 Case example two post genetic test Patient y was 5 yrs old when he required an appendicectomy because of acute appendicitis. During the operation he developed a progressive rise in heart rate, end-tidal CO2 and temperature. When his temperature was 39.1 C the Isoflurane was discontinued, dantrolene administered and the patient actively cooled. The procedure was completed using intravenous anaesthesia and y made an uneventful recovery. When the family was informed that y had suffered a serious complication of anaesthesia, y s grandmother volunteered the information that one of her cousin s children had suffered from malignant hyperthermia. Referral of the family to the MH Unit in Leeds revealed that the previous index case had been confirmed as MH susceptible by muscle biopsy 10 years previously and subsequent DNA analysis detected a heterozygous RYR1 variant c.487c>t, p.arg163cys which meets international consensus criteria for diagnostic use in MH susceptibility. Patient y was found to carry the same RYR1 variant. POST GENETIC TEST COSTS 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 Type of test Testing for known familial mutation Cost 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 170 170 37. Estimated savings between two case examples described 6,830
UKGTN Testing Criteria Test name: Malignant Hyperthermia Susceptibility 1 and 5 Approved name and symbol of disorder/condition(s): Malignant Hyperthermia, susceptibility to, 1 (MHS1) Malignant Hyperthermia, susceptibility to, 5 (MHS5) Approved name and symbol of gene(s): ryanodine receptor 1 (skeletal); RYR1 calcium channel, voltage-dependent, L type, alpha 1S subunit; CACNA1S OMIM number(s): 145600 601887 OMIM number(s): 180901 114208 Patient name: Patient postcode: Date of birth: NHS number: Name of referrer: Title/Position: Referrals will only be accepted from one of the following: Referrer Consultant Anaesthetist Consultant Clinical Geneticist Medical Officer, Institute of Naval Medicine (Military personnel) 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 Any one of the following: meets criteria Adverse reaction to general anaesthesia involving any combination of unexplained*: increase in carbon dioxide production, tachycardia, temperature increase, muscle rigidity, rhabdomyolysis, disseminated intravascular coagulation, death. Initial signs should be evident during anaesthesia or within 60 minutes of discontinuation of anaesthesia. Postoperative rhabdomyolysis and clinical exclusion of other myopathies Known case of malignant hyperthermia where no familial mutation identified Family history of malignant hyperthermia that has not been confirmed by muscle biopsy and/or where no familial mutation identified. Family history of unexplained perioperative death Exertional rhabdomyolysis or recurrent rhabdomyolysis where no cause identified following neurological work-up. Persistently raised serum creatine kinase concentration of unknown cause (idiopathic hyperckaemia). Other causes of raised creatine kinase should be excluded by neurologist.
Exertional heat stroke, especially in low-risk climatic conditions, where known predisposing factors have been excluded At risk family members where familial mutation is known. Additional Information: *The MH Unit in Leeds provides expert advice on the clinical features of malignant hyperthermia, none of which is specific for the condition. Of referrals of possible MH reactions made to the Unit, approximately 50% can be attributed to other causes and MH excluded on the basis of expert review of the clinical records. The MH Unit also maintains a national registry of MH cases and families and can advise on risk of family members and extent and findings of RYR1 and CACNA1S analyses in the family. 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.