rhabdomyolysis and metabolic myopathies 30 gene panel

Approval Date: March 2015 Submitting Laboratory: Sheffield RGC Copyright UKGTN © 2015 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 and symbol as published on the OMIM database (alternative names will be listed on the UKGTN website)

If this submission is for a panel test please complete appendix 1 listing all of the conditions included using approved OMIM name, symbol and OMIM number.

Panel test - please see appendix 1

2. OMIM number for disorder/condition

If a panel test – see 1. above


3a. Disorder/condition – please provide, in laymen’s terms, a brief (2-5 sentences) description of how the disorder(s) affect individuals and prognosis.

Rhabdomyolysis is the breakdown of skeletal muscle, typically manifesting as muscle pain, weakness and dark urine, with acute renal failure in severe cases; treatment and outcome depend on diagnosing the specific cause of the symptoms.

3b. Disorder/condition – if required please expand on the description of the disorder provided in answer to Q3a.

Rhabdomyolysis is a symptom that can have many causes, both acquired and genetic. Inherited metabolic myopathies are a significant cause, but with a wide range of causes including defects of glycogen storage, lipid metabolism and skeletal muscle channelopathies, as well as muscular dystrophies and mitochondrial disease. Exercise intolerance, muscle pain and weakness, and myoglobinuria are common symptoms that make a specific diagnosis very challenging due to the diversity of conditions. Treatments focus on progressive exercise training and condition-specific dietary management and supplements.

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.


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 appendix 1 listing all of the genes included using approved HGNC name, symbol, number and OMIM number.


6a. OMIM number(s) for gene(s)



6b. HGNC number(s) for gene(s)



7a. Gene – description(s)

If this submission is for a panel test, please provide total number of genes.

30 genes


7b. Number of amplicons to provide this test (molecular) or type of test (cytogenetic)

(n/a for panel tests)

N/A – panel test

7c. GenU band that this test is assigned to for index case testing.

Band G

8. Mutational spectrum for which you test including details of known common mutations

(n/a for panel tests) If this application is for a panel test to be used for different clinical phenotypes and/or various sub panel tests – please contact the team for advice before completing a Gene Dossier

N/A – panel test

9a. Technical method(s) – please describe the test.

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 MiSeq using the MiSeq Reagent Kit v2 performing 2 x 150 bp end paired reads.

Data Analysis

Bases on the open source ‘Best Practices’ workflow by the Broad Institute (for additional information, see http://www.broadinstitute.org/gatk/guide/best-practices).

BWA alignment of reads to human genome build hg19.

Generation of depth of coverage reports and checked using Alamut v 2.2 (Rev1) (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.

Identification of variants using HaplotypeCaller. Annotation from dbSNP and COSMIC (currently dbSNP138 and COSMIC v67 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 all 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.

Gaps in protein-coding regions and +/-25bp introns are covered by Sanger sequencing.

9c. Does the test include MLPA?

(For panel tests, please provide this information in appendix 1)



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.

Assay is provided by the laboratory.

11. Validation process

Please explain how this test has been validated for use in your laboratory or submit your internal validation documentation. If this submission is for a panel test, please provide a summary of evidence of:

i) instrument and pipeline validation, and

ii) panel verification for the test

Please submit as appendices to the Gene Dossier (these will be included in the published Gene Dossier available on the website).

Please note that the preferred threshold for validation and verification is 95% sensitivity with 95% Confidence Intervals.

Please refer to two attached validation documents.

12a. Are you providing this test already?

No – validation just completed.

12b(i). If yes, how many reports have you produced?

N/A

12b(ii). Number of reports mutation positive?

N/A

12b(iii). Number of reports mutation negative?

N/A

12b(iv). Please provide the time period in which these reports have been produced and whether in a research or a full clinical diagnostic setting.

N/A

13a. Is there specialised local clinical/research expertise for this disorder?

Yes 13b. If yes, please provide details

Dr R Quinlivan -Consultant in Neuromuscular Disease, MRC Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, London. Dr Quinlivan leads the NCG-funded National Diagnostic and Management Centre for McArdle Disease and related Disorders. Sheffield Diagnostic Genetics service was commissioned as part of this service to provide DNA testing for muscle GSDs.

Dr S Olpin – Consultant Clinical Scientist in Inherited Metabolic Disease, Sheffield Children’s NHS Foundation Trust – has a specialist interest in fatty acid oxidation disorders and diagnosis of rhabdomyolysis (Andresen et al (1999); Olpin et al (2003); Olpin et al (2005); Olsen et al (2007); Andresen et al (2012))

14. Based on experience what will be the national (UK wide) activity, per annum, for:

Index cases: 175

Estimate is based on 2013-2014 UK referrals for McArdle disease (both Sheffield and Birmingham Children’s), CPTII deficiency and VLCAD deficiency where no mutations were detected.


The estimated incidence calculated in section 17b is likely to be a significant underestimate, as incidence figures are available for only a very small number of these conditions, and this presentation is likely to consist of small numbers of a large variety of different disorders. In particular, RYR1 mutations are a recent, and currently under-reported cause of rhabdomyolysis/metabolic myopathies. As commented by Quinlivan and Jungbluth (2012 Dev Med Child Neurol 54:886-891) “Mutations in RYR1 are becoming increasingly recognized and may be an important cause of exercise intolerance and rhabdomyolysis”. However, there are no published data at present to quantify this. Preliminary results from the rhabdomyolysis research study being carried out by UCLH have shown that the most common finding so far has been mutations in the RYR1 gene.

Family members where mutation is known: 35 Assumes a clinical sensitivity of approximately 50%, and a 40% chance of a familial test being requested per diagnosis.

15. 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 laboratory has equipment and infrastructure in place to provide the full national need. However, the research-based panel test at the Institute of Neurology, Queen Square may be translated to a diagnostic panel. We anticipate collaborating with this group to agree a common panel based on the results of their research study and our diagnostic work.

16. 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


EPIDEMIOLOGY 17a. Estimated prevalence of conditions in the general UK population Prevalence is total number of persons with the condition(s) in a defined population at a specific time. Please identify the information on which this is based. For panel tests, please provide estimates for the conditions grouped by phenotypes being tested. UK population, mid-2012 = 63.7 million persons (Office for National Statistics) The diversity of inherited metabolic causes of rhabdomyolysis makes estimation of prevalence and incidence very difficult. Figures for the more common causes: adult-onset Pompe disease 1/148,000 (Martiniuk et al. 1998 Am J Med Genet 79:69-72); McArdle disease 1/100,000 (Haller et al. 2000 Arch Neurol 57:923-924); CPTII deficiency unknown (>300 myopathic cases reported (GeneReviews); estimate 1/100,000); VLCAD deficiency 1/85,000 (Lindner et al., J Inherit Metab Dis 35:269-277); central core disease (RYR1) 1/250,000 (Norwoord et al. 2009 132:3175-3186). Combined estimated prevalence = 1/26,000 Therefore estimated UK prevalence = 1/26,000 x 63,700,000 = 2450 persons 17b. Estimated annual incidence of conditions in the general UK population Incidence is total number of new cases in a year in a defined population. Please identify the information on which this is based. For panel tests, please provide for groups of conditions.

UK births, 2012 = 812,970 (Office for National Statistics) McArdle disease (~1 in 100,000) and CPTII deficiency (~1 in 300,000) represent the more common causes of exercise-induced rhabdomyolysis (van Adel et al 2009 J Clin Neuromusc Dis 10:97-121). The adult onset form of Pompe disease has an estimated incidence of 1/57,000 (Ausems et al. 1999 Eur J Hum Genet 7:713–6). VLCAD deficiency 1/50,000 (Quinlivan and Jungbluth 2012 Dev Med Child Neurol 54:886-891); central core disease (RYR1) unknown. Combined estimated incidence = 1/20,000 Therefore, estimated UK incidence = 1/20,000 x 812,970 = 41 new cases per year 18. Estimated gene frequency (Carrier frequency or allele frequency) Please identify the information on which this is based. n/a for panel tests.

N/A – panel test

19. Estimated penetrance of the condition. Please identify the information on which this is based n/a for panel tests

N/A – panel test

20. Estimated prevalence of conditions in the population of people that will be tested. n/a for panel tests.

N/A – panel test

INTENDED USE (Please use the questions in Annex A to inform your answers)

21. 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 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


TEST CHARACTERISTICS 22. 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 note that the preferred threshold for validation and verification is 95% sensitivity with 95% Confidence Intervals.

Validation data for the Miseq analysis pipeline and GSD SureSelect panel shows that the test sensitivity is >99% (+/-0.08 at 95% confidence level) and test specificity is >99% compared to Sanger sequencing.

The NGS panel would not currently detect heterozygous partial or whole gene deletions or detect deep intronic mutations within the genes tested (estimated to account for <2% mutation alleles).

23. 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 two years service.

Sensitivity is likely to be limited by the diversity of causes, including mitochondrial/respiratory chain defects which are covered by a nationally funded service and therefore not included in the panel. Sensitivity can be improved by use of a diagnostic algorithm (e.g. Quinlivan and Jungbluth 2012 Dev Med Child Neurol 54:886-891).

A research study is currently in progress at the Institute of Neurology, Queen Square using targeted panels and exome sequencing to identify genetic causes of rhabdomyolysis. Preliminary results gave an estimated sensitivity of 52%. The final results will inform clinical sensitivity and may also modify the genes included in the panel. Any changes to the panel test would require notification to UKGTN for further consideration.

Clinical specificity should be close to 99% (in house validation data showing no false positives). Variants of uncertain clinical significance will be found in a small proportion of cases.

24. 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 – panel test

25. 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.

n/a for panel tests

N/A – panel test


CLINICAL UTILITY 26. How will the test change the management of the patient and/or alter clinical outcome? Please describe associated benefits for patients and family members. If there are any cost savings AFTER the diagnosis, please detail them here.

Accurate molecular genetic diagnosis of the underlying metabolic condition enables specific clinical management of the condition. For glyco(geno)lytic disorders, careful management of aerobic exercise patterns and creatine supplementation is beneficial. For fatty acid oxidation disorders, a fat-reduced, medium-chain triglyceride-supplemented diet, avoidance of fasting and avoidance of prolonged exercise is the basis of treatment/management, with an emergency regimen (including intravenous dextrose) in the case of a metabolic decompensation. Definitive and rapid carrier, pre symptomatic, perinatal, prenatal or preimplantation testing for affected families is also enabled.

The availability of a panel test will result in a significant cost saving due to the removal of the need for sequential gene testing, enzymology and possible associated need for tissue biopsy and will lead to a much quicker diagnosis for the patient.

It will also clarify the inheritance pattern and therefore the associated recurrence risks for the patient’s wider family.

27. If this test was not available, what would be the consequences for patients and family members?

The lack of panel testing would mean the continued need for investigations including a possible tissue (muscle, skin) biopsy, which would be potentially distressing and costly for the patient. There would be a longer time to diagnosis, or potentially no diagnosis made, and in some instances there is a risk of misdiagnosis leading to sub-optimal treatment of the underlying metabolic disorder. Sub-optimal management of therapy, as a result of an incorrect or non-specific diagnosis, may result in continuing avoidable morbidity.

28. 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.

Many definitive biochemical investigations would require cultured fibroblasts; molecular testing removes the need for invasive tissue biopsy and costs associated with tissue culture.

Enzymatic testing is available for some conditions; however these can be cumulatively expensive and have been known to give non-specific, inconclusive or non-reproducible results.

29a. What unexpected findings could this test show? 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.

Heterozygous mutations in the RYR1 gene can also be associated with malignant hyperthermia susceptibility. This could also impact on family members.

29b. Please list any genes where the main phenotype associated with that gene is unrelated to the phenotype being tested by the panel.

N/A

30. If testing highlights a condition that is very different from that being tested for, please outline your strategy for dealing with this situation.

Any unusual findings, once confirmed, would be reported to the referring clinician, but with additional direct discussions, and, where appropriate, a strong recommendation to refer the individual/family to the clinical genetics service.


31. If a panel test, is this replacing an existing panel/multi gene test and/or other tests currently carried out through UKGTN using Sanger sequencing? If so, please provide details below.

No.

The panel does overlap with the following Sheffield tests currently on UKGTN: -

- CPTII deficiency (CPT2 gene)

- Carnitine acyl-carnitine translocase deficiency (SLC25A20 gene)

- Glycogen storage diseases (Glycogen Storage Disease 32 Gene Panel approved 2014)

However, referral routes/categories/symptoms can vary widely, and so ideally these separate tests would still be listed and offered.

32. Please describe any specific ethical, legal or social issues with this particular test.

N/A IS IT A REASONABLE COST TO THE PUBLIC? 33. In order to establish the potential costs/savings that could be realised in the diagnostic care pathway, please list the tests/procedures that would be required in the index case to make a diagnosis if this genetic test was not 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)

Glycogen enzyme screen Fibroblast cell culture Fatty acid oxidation studies Sanger sequencing

£ 468 £ 250 £ 275 £ 600

Costs and types of physiological tests (e.g. ECG) Cost and types of other investigations/procedures (e.g. biopsy) muscle biopsy

skin biopsy £ 292 £ 126

Total cost of tests/procedures no longer required (please write n/a if the genetic test does not replace any other tests procedures in the diagnostic care pathway)

£2011


34. Based on the expected annual activity of index cases (Q14), please calculate the estimated annual savings/investments based on information provided in Q33.

Number of index cases expected annually 175 Cost to provide tests for index cases if the genetic test in this Gene Dossier was not available (see Q32)

£2011

Total annual costs pre genetic test £351,925 Total annual costs to provide genetic test £166,250 Additional savings for 100% positive rate for index cases

£185,675

Percentage of index cases estimated to be negative

50%

Number of index cases estimated to be negative 88 Costs to provide additional tests for index cases testing negative

88 x £1411 = £124,168 as no need to repeat sanger tests

Total savings for tests for index patient activity £61,507 Total costs for family members £105 x 35 = £3675 If there is a genetic test already available and some of the family testing is already being provided, please advise the cost of the family testing already available

£105 x 10 = £1050

Total costs for family members minus any family member testing costs already provided

£2625

Additional savings for all activity expected in a year

£58,882 savings

35. REAL LIFE CASE STUDY Please provide a case study that illustrates the benefits of this test A 28-year-old Turkish male was referred to the Neuromuscular Centre at National Hospital for Neurology and Neurosurgery in London with exercise-induced muscle pain and cramps. Symptoms had been present since early childhood. He was able to jog slowly, but he had never been able to run quickly for more than a few minutes without stopping due to myalgia and cramps. He also developed similar symptoms when walking quickly or up an incline. Since the age of 17 years, he had a number of episodes of “coca-cola” coloured urine consistent with myoglobinuria following exercise. After playing football for a short period of time, he developed myalgia and weakness in his lower back and proximal lower limb muscles accompanied by dark urine. On one occasion, after playing football, he required admission to hospital for intravenous fluids because his CK level was found to be significantly elevated at 75,000 IU/L (normal <250 IU/L). His parents were first cousins. His neurological examination revealed mild left-sided scapular winging. His BMI was 25.8 kg/m2 and he walked 1000 metres during a twelve minute walk test (TMWT) without evidence of a second-wind. Investigations revealed a normal serum CK at rest, serum carnitine and acylcarnitines, white cell α-glucosidase activity and co-enzyme Q10, serum amino acid profile and urinary organic acids levels. In vitro analysis using cultured skin fibroblasts showed normal fatty acid oxidation. The patient underwent a muscle biopsy of vastus lateralis after informed consent. Morphological studies showed no evidence of vacuoles on haematoxylin and eosin and Gomori-trichrome stained sections or cores with oxidative stains. Glycogen, lipid and mitochondrial staining, with periodic acid-Schiff (PAS), Sudan Black and sequential cytochrome oxidase and succinate dehydrogenase stains respectively, were unremarkable. Acid phosphatase staining and glycolytic histochemical stains for phosphorylase and phosphofructokinase were normal. Routine immunohistochemistry for the dystrophin panel of staining was normal. Electron microscopy analysis revealed a mild increase in muscle glycogen. Biochemical studies on muscle homogenate revealed normal CPT II, AMPD and respiratory chain enzymes activities. Muscle glycolytic enzymes activities as phosphorylase b kinase, phosphorylase, phosphofructokinase and phosphoglycerate mutase, phophoglycerate kinase, phosphoglucomutase were normal.


Genetic investigations were undertaken, but no mutations were detected in RYR1, LPIN1, PYGM or PGAM2, using Sanger sequencing (combined cost ~£2300). A sample of genomic DNA from the individual was then run on the rhabdomyolysis panel. Analysis of the ENO3 gene revealed a homozygous variant c.559G>A located in exon 7 affecting a highly conserved amino acid, changing a glutamic acid residue to lysine (p.Glu187Lys). The diagnosis was confirmed enzymatically: β-enolase activity was markedly reduced, 0.06 (10% ) (n.v. 0.66±0.1). Parents were confirmed as heterozygous carriers. If the panel test had been available and used early in the diagnostic pathway, the diagnosis could have been made much sooner, without the need for skin and muscle biopsies, and with significant reduced costs for histochemical and enzymatic testing. The cost of genetic testing would also have been reduced by £1350. A single enzymatic assay on a blood sample would then have confirmed the genetic findings.

TESTING CRITERIA

36. Please only complete this question if there is previously approved Testing Criteria.

Please contact the UKGTN office if you are unsure whether testing criteria is available.

36a. Do you agree with the previously approved Testing Criteria? Yes/No

36b. If you do not agree, please provide revised Testing Criteria on the Testing Criteria form and explain below the reasons for the changes.


UKGTN Testing Criteria Test name: Rhabdomyolysis and Metabolic Myopathies 30 Gene Panel

Approved name and symbol of disorder/condition(s): See website listing

OMIM number(s):

Approved name and symbol of gene(s): See website listing

OMIM number(s):

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 Paediatrician Consultant Neurologist Consultant in Metabolic Medicine Consultant Clinical Geneticist Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Tick if this patient

meets criteria Myalgia, muscle weakness, cramps, myoglobinuria, triggered by exercise/heat/fasting/infection AND

Suggestive blood/urine tests: acylcarnitines, lactate, amino acids, organic acids, Creatine Kinase (CK) AND

Mitochondrial myopathy/dystrophinopathy unlikely/excluded AND Common McArdle disease (PYGM)/CPTII deficiency (CPT2) mutations excluded, as appropriate

Additional Information: 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.


Diagnostic algorithm proposed by Drs R Quinlivan and E Murphy as part of an A3 change proposal to NHS England.

Approval Date: March 2015 Submitting Laboratory: Sheffield RGC

Copyright UKGTN © 2015 14


Appendix 1 Genes in panel test and associated conditions. Rows highlighted in yellow show where the genes were being fully analysed in the context of a single separate UKGTN test at time of submission. HGNC standard name and symbol of the gene

HGNC number

OMIM number

OMIM standard name of condition and symbol

Mode of inheritance

OMIM number

Evidence of association between gene(s) and condition

% of horizontal coverage of gene

MLPA Comments

glucosidase, alpha; acid; GAA

4065 606800 Glycogen storage

disease II

AR 232300

GAA mutations found on 95% of alleles in 29 GSD II patients

(Hermans et al, 2004); 197 different pathogenic mutations in GAA have been described

(Kroos et al, 2008).

100% No

amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase;

AGL


disease III AR 232400

AGL mutations found in 96% of alleles in 68 GSD III patients

(Goldstein et al, 2010). 100% No

glucan (1,4-alpha-), branching enzyme 1;

GBE1


disease IV AR 232500

GBE mutations found in 100% of alleles in 8 GSD IV patients

(Bruno et al, 2004); 37 different pathogenic mutations in GBE1 have been described

(Li et al, 2010).

100% No

phosphorylase, glycogen, muscle;

PYGM


disease V AR 232600

PYGM mutations found on 74% of alleles in 40 GSD V

patients (Tsujino et al, 1993); PYGM mutations found on 92% of alleles in 62 GSD V patients (Bruno et al, 2006).

100% No

phosphofructokinase, muscle; PFKM


disease VII AR 232800

18 PFKM mutations found in 9 Ashkenazi Jewish families

affected with GSD VII (Sherman et al, 1994); at least

15 mutations in PFKM have been reported (Nakijima et al,

2002).

100% No

phosphorylase kinase, alpha 1 (muscle);


disease IXd; GSD9D XR 300559

Case report of a male patient with myopathy and absent

100% No


PHKA1 phosphorylase kinase activity with a frameshift mutation in PHKA1 (Wuyts et al, 2005);

1/9 patients with Phk deficiency and glycogenosis found to have a mutation in

PHKA1 (Burwinkel et al, 2003).

phosphorylase kinase, gamma 1 (muscle);

PHKG1 8930 172470 NK NK NK

No mutations in PHKG1 have been identified to date. It is

included as a candidate gene as it is the muscle homologue

of the PHKG2 gene associated with GSD type IX

100% No

fructose-1,6-bisphosphatase 2;

FBP2 3607 603027 NK NK NK

No mutations in FBP2 have been identified to date. It is

included as a candidate gene as it is the muscle homologue of the FBP1 gene associated

with FBP1 deficiency

100% No

glycogen synthase 1 (muscle); GYS1

4706 138570 Glycogen storage disease 0, muscle

AR 611556

3 siblings with GSD 0 found to be homozygous for mutation in

GYS1 (Kollberg et al, 2007); Homozygous GYS1 deletion

identified in patient with muscle-specific glycogen

synthase deficiency (Cameron et al, 2009)

100% No

phosphoglycerate kinase 1; PGK1

8896 311800 Phosphoglycerate kinase 1 deficiency

XR 300653

Mutation in PGK1 identified in a 27-year-old Japanese male with PGK1 deficiency (Fujii et

al, 1992); 20 different mutations in PGK1 have been

identified and functional studies used to correlate with

pathological outcome (Chiarelli et al, 2012)

100% No

glycogenin 1; GYG1 4699 603942 Glycogen storage

disease XV; GSD15 AR 613507

Deficiency of glycogenin-1 seen in a patient with glycogen depletion and caridomyopathy

and found to be compound heterozygous for mutations in

GYG1. Neither mutation observed in 200 control

100% No


chromosomes (Moslemi et al, 2010)

phosphoglycerate mutase 2 (muscle);

PGAM2 8889 612931

Glycogen storage disease X; GSD X

AR 261670

In 5 patients with muscle phosphoglycerate mutase deficiency, Tsujino et al.

(1993) identified 3 homozygous or compound

heterozygous mutations in the PGAM2 gene.

100% No

aldolase A, fructose-bisphosphate; ALDOA


disease XII; GSD12 AR 611881

Case report of one patient with aldolase A deficiency found to be homozygous for a mutation

in ALDOA (Kreuder et al, 1996); Case report of one

patient with aldolase A deficiency compound

heterozygous for mutations in ALDOA (Yao et al, 2004);

100% No

enolase 3 (beta, muscle); ENO3


disease XIII; GSD13 AR 612932

Case report of one patient with a severe muscle enolase

deficiency found to be compound heterozygous for mutations in ENO3 (Comi et

al, 2001)

100% No

lactate dehydrogenase A; LDHA


disease XI; GSD11 AR 612933

18 individuals from four Japanese families affected

with LDHA deficiency found to be homozygous for the same

20bp deletion in LDHA (Maekawa et al 1991);

100% No

phosphoglucomutase 1 PGM1

8905 171900

Congenital disorder of glycosylation, type It;

Glycogen storage disease type IV

AR 614921 612934

One patient with exercise intolerance and episodic

rhabdomyolysis found to be compound heterozygous for mutations in the PGM1 gene (Stojkovic et al (2009)). Two

unrelated patients with congenital disorder of

glycosylation type It were found to have 2 different

homozygous mutations in the PGM1 gene (Timal et al

(2012))

100% No

RanBP-type and 15864 610924 RBCK1 deficiency AR NK 10 individuals with myopathy 100% No


C3HC4-type zinc finger containing 1

RBCK1

and cardiomyopathy and accumulation of polyglucosan

bodies in affected tissues were found to be homozygous or compound heterozygous for

nonsense or truncating mutations in RBCK1 (Nilsson

et al 2013)

acyl-CoA dehydrogenase, very long chain; ACADVL

92 609575

Acyl-CoA dehydrogenase, very long chain, deficiency

of; ACADVLD

AR 201475

9 different ACADVL mutations observed in 4 patients with

VLCAD deficiency (Andresen et al, 1996); 58 different

ACADVL mutations observed in 55 patients with VLCAD deficiency (Andresen et al,

1999)

100% No

carnitine palmitoyltransferase 2;

CPT2 2330 600650

CPT II deficiency, lethal neonatal

CPT II deficiency,

infantile

CPT II deficiency, late-onset

AR

608836 600649 255110

8 patients with CPTII deficiency found to be

homozygous for common mutation in the CPT2 gene

(Taroni et al 1993); mutations found on 37/38 alleles in

patients with CPTII deficiency (Thuillier et al, 2003)

100% No

carnitine palmitoyltransferase 1B (muscle); CPT1B

2329 601987 NK NK NK

No mutations in CPT1B have been identified to date. It is

included as a candidate gene as it is the muscle homologue of the CPT1A gene associated

with CPT1 deficiency

100% No

solute carrier family 22 (organic

cation/carnitine transporter), member

5; SLC22A5

10969 603377

Carnitine deficiency,

systemic primary; CDSP

AR 212140

Affected individuals from 3 families found to carry

mutations in SLC22A5 on both alleles (Nezu et al, 1999); 61

cases of primary carnitine deficiency reported in

literature; 111 disease-causing mutations in SLC22A5

reported (Shibbani et al, 2013).

100% No

hydroxyacyl-CoA dehydrogenase/3-

4801 600890 Long chain 3-

hydroxyacyl-CoA AR

609016

Common mutation in HADHA accounts for ~87% of LCHAD

100% No


ketoacyl-CoA thiolase/enoyl-CoA

hydratase (trifunctional protein), alpha

subunit; HADHA

dehydrogenase deficiency

Trifunctional protein

deficiency

609015

deficiency mutations (Ijlst et al, 1996); Mutations in HADHA

and HADHB were detected in 100% of alleles from patients

with Trifunctional Protein Deficiency (45/52 HADHA;

7/52 HADHB) (Boutron et al, 2011)

hydroxyacyl-CoA dehydrogenase/3-

ketoacyl-CoA thiolase/enoyl-CoA

hydratase (trifunctional protein), beta subunit;

HADHB

4803 143450

Trifunctional protein

deficiency

AR 609015

2 patients with Trifunctional protein deficiency found to have mutations in HADHB

(Ushikobo et al, 1996); Mutations in HADHA and HADHB were detected in

100% of alleles from patients with Trifunctional Protein

Deficiency (45/52 HADHA; 7/52 HADHB) (Boutron et al,

2011)

100% No

electron-transfer-flavoprotein, alpha polypeptide; ETFA

3481 608053 Multiple acyl-CoA dehydrogenase

deficiency; MADD AR 231680

One patient with MADD found to homozygous for mutation in

ETFA (Indo et al, 1991); Mutations in ETFA, ETFDH or ETFB found on 93% of alleles in 15 Japanese patients with

MADD (Yotsumoto et al, 2008)

100% No

electron-transfer-flavoprotein, beta

polypeptide; ETFB 3482 130410

Multiple acyl-CoA dehydrogenase


Mutations in ETFB identified in 2 brothers with GA2 (Colombo

et al, 1994); Mutations in ETFA, ETFDH or ETFB found

on 93% of alleles in 15 Japanese patients with GA2

(Yotsumoto et al, 2008)

100% No

electron-transferring-flavoprotein

dehydrogenase; ETFDH

3483 231675 Multiple acyl-CoA dehydrogenase


Mutations in ETFDH found on both alleles in 4 patients with

MADD (Liang et al, 2009); Mutations in ETFA, ETFDH or ETFB found on 93% of alleles in 15 Japanese patients with GA2 (Yotsumoto et al, 2008)

100% No

Lipin 1; LPIN1 13345 *605518 Myoglobinuria, acute recurrent, autosomal recessive

AR #268200 Zeharia et al (2008) Mutations detected in 5 unrelated families with recurrent rhabdomyolysis.

100% No


Michot et al (2012) Mutations detected in 18/171 individuals with muscle symptoms of varying severity.

ryanodine receptor 1 (skeletal); RYR1

10483 *180901 Central core disease of muscle

AD/AR #117000 Wappler et al (2001) Single mutations detected in 3/11 patients with exercise-induced rhabdomyolysis, screening for known mutations only. Sambuughin et al (2009) RYR1 mutations detected in 5/6 unrelated African American men with exercise-induced rhabdomyolysis.

100% No

Iron-sulfur cluster assembly enzyme; ISCU

29882 *611911 myopathy with lactic acidosis, herditary

AR #255125 Mochel et al (2008) Homozygous deep intronic mutation found in 3 Swedish patients with myopathy and lactic acidosis. Kollberg et al (2009) 2 brothers with muscle wekness, exercise intolerance and cardiomyopathy found to be compound heterozygous for Swedish mutation and a missense change inherited from Finnish mother.

100% No

Caveolin 3; CAV3 1529 *601253 Myopathy, distal, Tateyama type Rippling muscle disease

AD/AR #614321 #606072

Gonzalez-Perez et al (2009) Spanish family with distal myopathy found to have a dominant missense mutation. Kubisch et al (2003) 2 unrelated individuals with muscle stiffness/weakness found to be homozygous for CAV3 mutations.

100% No

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Sheffield Children’s NHS Foundation Trust Department: Sheffield Diagnostic Genetics Service

Title: General: Protocol/Service validation form

Document reference number: 401.208

Date of issue: 19.06.2014 Version number: 2

Author Darren Grafham

Authorised by: Jennifer Dawe

Page 1 of 1

General: Protocol Validation Form SOP

Complete this form electronically, save in G:\BUSINESS\PROTOCOLS\VALIDATION along with the Document No. and Name (Drive N for Cytogenetics staff) Brief description of reason for new or revised procedure Validation of new next generation sequencing (NGS) Sureselect panels for a number of genes associated with - fatty acid metabolism disorders - peroxisome disorders - hyperammonaemia disorders - rhabdomyolysis. Sequencing data is derived from the Illumina MiSeq sequencing platform and analysed using the ‘Kevin’ analysis pipeline. The MiSeq platform and ‘Kevin’ analysis pipeline have been validated previously using two SureSelect gene panels and one Trusight gene panels (see Validation of analysis pipeline and depth of coverage.pdf). List all SOPs to be varied 401.058 Complete analysis of raw next generation sequencing data, including alignment, QC, variant-calling, and report-generation Describe variations to be tested along with relevant SOP No. Ensure that the analysis of next generation sequencing data produced using the new IEM Sureselect probe panel (ELID 0660881) correlates with variants previously identified by either Sanger sequencing or by NGS using a previously validated Sureselect probe panel for a number of genes associated with glycogen storage disorders (ELID 0423281). Determine the depth of coverage over the genomic regions targeted by the IEM SureSelect probe panel, and identify regions or gaps where coverage is reproducibly low for all samples. Has test variation been discussed and approved by Head of Service? Y

Initialled proposer -

Liz Allen

Initialled Head of Service -

Richard Kirk

Start date of test 02/06/2014 End date of test 16/07/2014







Page 2 of 1

Results: Results are summarised in the following appendix. Conclusions: All variants previously identified by Sanger sequencing or by NGS using an alternative, validated SureSelect probe set were identified using the IEM NGS panel. 118 concordants variants were detected over a total of 28 genes; no discordant variant calls were detected. Depth of coverage data was examined over the genomic intervals targeted by the IEM SureSelect probes. Depth of coverage was reproducibly high: (97.5 – 98.8% at 30 x coverage or above). Fifteen intronic or exonic regions with a depth of coverage of less than 30 x were seen in all 6 samples where coverage was analysed in detail. Nine of these regions are located in introns or the 5’ untranslated region of the particular gene, regions which are thought to be less likely to harbour pathogenic mutations. Conclusions reviewed and verified by Head of Service

R J Kirk

Conclusions reviewed and verified by Head of lab services

D Grafham

Further testing required? Y / N Alteration to existing SOP/SOPs recommended Y / N If yes, specify recommended changes and SOP No. New SOPs for 4 panels required. Changes added to SOP by; initials RJK Date 26/07/2014 NB – Return form to Quality Lead







Page 3 of 1

Appendix Validation of new next generation sequencing (NGS) IEM Sureselect panels A SureSelect custom probe library has been designed for a total of 80 genes associated with various IEM disorders (the ‘IEM panel’). The SureSelect probes were designed to all coding exons +/-25bp into intronic regions for all genes, with additional intronic regions being included in the design if mutations had previously been reported in the Human Gene Mutation Database (http://www.biobase-international.com/product/hgmd). The genes have been separated into four clinically relevant sub-panels in order to aid analysis of data: 1) Fatty acid metabolism disorders

Condition Gene ACAD9 deficiency (complex I deficiency) ACAD9 Beta-ketothiolase deficiency ACAT1 Brown-Vialetto-van Laere syndrome 1 (C20ORF54) SLC52A3 Brown-Vialetto-van Laere syndrome 2 SLC52A2 CACT deficiency SLC25A20CPTI deficiency CPT1A CPTI deficiency (muscle) CPT1B CPTII deficiency CPT2 HADH (SCHAD) deficiency HADH MADD ETFA MADD ETFB MADD (riboflavin-responsive) ETFDH MCAD deficiency ACADM Mitochondrial HMG-CoA lyase deficiency HMGCL Mitochondrial HMG-CoA synthase deficiency HMGCS2 Mitochondrial TFP deficiency HADHA Mitochondrial TFP deficiency HADHB Riboflavin deficiency SLC52A1 SCAD deficiency (??secondary only??) ACADS SCOT deficiency OXCT1 Systemic primary carnitine deficiency (carnitine transporter) SLC22A5 VLCAD deficiency ACADVL







Page 4 of 1

2) Peroxisome disorders

Condition Gene Adult Refsum disease PHYH Alpha-methylacyl-CoA racemase (AMACR) deficiency AMACR D-Bifunctional protein deficiency HSD17B4 Encephalopathy, lethal, due to defective mitochondrial peroxisomal fission DNM1L Leukoencephalopathy with dystonia and motor neuropathy SCP2 Peroxisomal Acyl-CoA oxidase deficiency ACOX1 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX1 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX6 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX10 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX12 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX26 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX13 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX14 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX16 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX19 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX3 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX5 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX2 Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum PEX11B Rhizomelic chondrodysplasia punctata type 1 (RCDP1) PEX7 Rhizomelic chondrodysplasia punctata type 2 (RCDP2) (DHAPAT deficiency) GNPAT Rhizomelic chondrodysplasia punctata type 3 (RCDP3) AGPS Type I primary hyperoxaluria (HP1) AGXT X-linked adrenoleukodystrophy ABCD1

3) Hyperammonaemia disorders

Condition Gene Arginase deficiency (hyperargininemia) ARG1 Argininosuccinic aciduria ASL Carbamoylphosphate synthetase I (CPS1) deficiency CPS1 Citrullinaemia type 2 (citrin deficiency) SLC25A13 Citrullinemia type I ASS1 HHH syndrome SLC25A15 Hyperinsulinism-hyperammonemia syndrome GLUD1 Lysinuric protein intolerance SLC7A7 MMA (mutase) MUT N-acetyl glutamate synthetase (NAGS) deficiency NAGS Ornithine aminotransferase (OAT) deficiency OAT Ornithine transcarbamylase (OTC) deficiency OTC PA PCCA PA PCCB







Page 5 of 1

4) Rhabdomyolysis/metabolic myopathies

Condition Gene Autosomal recessive recurrent acute myoglobinuria LPIN1 CARNITINE PALMITOYLTRANSFERASE I, MUSCLE* CPT1B * Carnitine palmitoyltransferase II (CPT II) deficiency* CPT2 * Central Core Disease RYR1 Glycogen storage disease 0, muscle GYS1 Glycogen storage disease II GAA Glycogen storage disease III AGL Glycogen storage disease IV / Polyglucosan body disease, adult form GBE1 Glycogen storage disease VII PFKM Glycogen storage disease X PGAM2 Glycogen storage disease XI LDHA Glycogen storage disease XII ALDOA Glycogen storage disease XIII ENO3 Glycogen storage disease XIV PGM1 Glycogen storage disease XV GYG1 Glycogen storage disease, type V; McArdle disease PYGM GSD Ixd; muscle phosphorylase kinase deficiency PHKA1 Hereditary myopathy with lactic acidosis ISCU Mitochondrial TFP deficiency* HADHA * Mitochondrial TFP deficiency* HADHB * MADD ETFA MADD ETFB MADD (riboflavin-responsive) ETFDH Muscular dystrophy, limb-girdle, type IC; Myopathy, distal, Tateyama type; Rippling muscle disease CAV3 Phosphoglycerate kinase 1 deficiency PGK1 RBCK1 deficiency RBCK1 Systemic primary carnitine deficiency (carnitine transporter)* SLC22A5 * very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency* ACADVL * No disease association to date FBP2 No disease association to date PHKG1

* gene also included in fatty acid metabolism disorder subpanel A total of 11 previously analysed patient samples were sequenced using the IEM SureSelect panel. Next generation sequencing data was generated and analysed using a previously validated MiSeq sequencing platform and the ‘Kevin’ analysis pipeline which utilises open source, industry standard packages based on the best practice guidance of the Broad Institute.







Page 6 of 1

Comparison with Sanger sequencing 1) Fatty acid metabolism disorders Sanger sequencing data was available for 3 patient samples over 3 genes within the fatty acid metabolism disorder subpanel.

Gene No. concordant variants No. discordant variants CPT2 1 0

ACADVL 5 0

ACADM 10 0

2) Peroxisome disorders Sanger sequencing data was available for 2 patient samples over 3 genes within the peroxisome disorders panel.

Gene No. concordant variants No. discordant variants PEX1 12 0

PEX6 11 0

PEX10 4 0

3) Hyperammonaemia disorders Sanger sequencing data was available for 3 patient samples over 3 genes within the hyperammonaemia disorders panel.

Gene No. concordant variants No. discordant variants ASS1 5 0

CPS1 16 0

NAGS 3 0

4) Rhabdomyolysis/metabolic myopathies Sanger sequencing data was available for 3 patient samples over 2 genes within the rhabdomyolysis panel. The data for 2 of these samples, for the ACADVL gene, has also been included in the validation data set for the fatty acid metabolism disorders

gene No. concordant variants No. discordant variants ACADVL 5 0

PFKM 1 0







Page 7 of 1

Comparison with NGS sequencing using a previously validated probe set - rhabdomyolysis panel. Some genes included within the rhabdomyolysis sub-panel are already included within a previously validated GSD SureSelect panel (see gene dossier #198). NGS data generated with the IEM SureSelect panel was compared to previous NGS data generated using the GSD SureSelect panel. Data was available for 2 patient samples over 10 genes within the rhabdomyolysis panel.

gene No. concordant variants No. discordant variants AGL 14 0

ALDOA 1 0

ENO3 3 0

GAA 16 0

GBE1 3 0

GYG1 1 0

GYS1 1 0

LDHA 4 0

PFKM 6 0

PHKG1 1 0

Summary of variant comparison data In total, 118 variants have been found to correlate between NGS data generated using the IEM SureSelect panel and data previously generated either by Sanger sequencing or NGS data generated using the GSD SureSelect panel. This data has been generated from 11 patient samples and covers 18 genes. No false negatives or positives were detected in the IEM panel NGS data.







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Depth of Coverage Previous in–house validation of the MiSeq sequencing platform and ‘Kevin’ analysis pipeline demonstrated that a read depth equal to or greater than 30x gave a detection rate greater than 99.9%. Therefore a depth of coverage of 30x has been selected as a cut-off for diagnostic sequencing. Below is a summary of the % of bp targeted by the SureSelect panel which shows a depth of coverage equal to or greater than 30x for all validation samples. For four samples the data was analysed further in order to determine the number of gaps, ie. exonic or intronic regions with depth of coverage significantly below 30 x.

Sample no % over 30 x coverage No. gaps 1 97.5 372 98.4 223 98.5 164 98.5 175 98.8 N/A6 98.2 N/A7 98.3 N/A8 98.7 N/A9 98.3 N/A

10 98 N/A11 97.6 N/A

Average 98.3 N/A N/A = not analysed 11 exonic or intronic regions gave a depth of coverage of below 30 x in all samples analysed. They were:

PGM1_Exon1 LPIN1_NM_145693.2_exon1 AGPS_exon_1 CPS1_intron_35 MUT_intron11 OAT_intron2 CPT1A_Exon_1 PEX5_exon_1 PFKM_Ex4_5pUTR RYR1_exon91 RYR1_intron101

Seven of these regions (highlighted) are located within introns or the 5’ untranslated region of the relevant gene and therefore are would be of less significance for detection of pathogenic mutations. All regions identified as being below 30 x on NGS analysis will be analysed by Sanger sequencing.

rhabdomyolysis and metabolic myopathies 30 gene panel

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