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
1. Disorder/condition – approved name and
symbol as published on the OMIM
database (alternative names will be listed on the
UKGTN website)
2. OMIM number for disorder/condition
Approved: Sept 2013
Malignant Hyperthermia, susceptibility to, 1 (MHS1)
Malignant Hyperthermia, susceptibility to, 5 (MHS5)
(Note, malignant hyperthermia is also known as
malignant hyperpyrexia)
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.
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.
3b Disorder/condition – if required please
expand on the description of the disorder
provided in answer to Q3a.
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.
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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)
32 long PCR fragments (23 for RYR1 and 9 for
CACNA1S) to include all exons and intronic
boundaries.
7c. GenU band that this test is assigned to
for index case testing
G (50-100 amplicons)
8. Mutational spectrum for which you test
including details of known common
mutations
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
5. Gene – approved name(s) and symbol as
published on HGNC database (alternative
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.
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
ii) Does the test include MLPA?
No
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
NGS
12a. Are you providing this test already?
Yes
Provided by the lab
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).
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.
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
this service?
14a. Is there specialised local
clinical/research expertise for this
disorder?
14b. If yes, please provide details
See 12b above
No
x Yes
The Leeds MH Investigation Unit has been the
national referral centre since 1971. With more than
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
15. Are you testing for other
genes/disorders/conditions closely allied
to this one? Please give details
16. Based on experience what will be the
national (UK wide) activity, per annum, for:
16a. Index cases
16b. Family members where mutation is
known
17a. Does the laboratory have capacity to
provide the expected national activity?
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
75
100
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
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
EPIDEMIOLOGY
19a. Estimated prevalence of condition in
the general UK population
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.
19b. Estimated incidence of condition in
the general UK population
Please identify the information on which this is
based
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.
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
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.
22. Estimated prevalence of condition in
the population of people that meet the
Testing Criteria.
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%.
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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
(n/a for panel tests)
Yes
No
Carrier testing for family members (n/a for panel tests)
Yes
No
Prenatal testing
Yes
No
Presymptomatic testing
(n/a for panel tests)
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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%
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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.
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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.
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)
Total cost tests/procedures no longer required
Type of test
Cost (£)
Open
muscle
biopsy with in
vitro
muscle
contracture tests
on the excised
specimens
and
histopathological
examination
3,500
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
Cost to provide tests for index cases if the
genetic test in this gene dossier was not
available (see Q34)
Total annual costs pre genetic test
Total annual costs to provide genetic test
Total savings
75
3,500
262,500
39,750
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.
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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
Cost
2 x Open muscle
biopsy with in vitro
muscle contracture
tests on the excised
specimens
and
histopathological
examination
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
Cost
Testing for known
familial mutation
170
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
37. Estimated savings between two case examples described £6,830
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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:
Date of birth:
Patient postcode:
NHS number:
Name of referrer:
Title/Position:
Lab ID:
Referrals will only be accepted from one of the following:
Referrer
Tick if this refers to
you.
Consultant Anaesthetist
Consultant Clinical Geneticist
Medical Officer, Institute of Naval Medicine (Military personnel)
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.
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013
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.
Approval Date: Sept 2013
Submitting Laboratory: Glasgow RGC
Copyright UKGTN © 2013