Plant sample testing for atypical myopathy

We offer testing of seeds, leaves and seedlings for the Hypoglycin A toxin known to cause atypical myopathy and these do not need to pre-booked with us. To find out if plant samples on your property contain the toxin owners can submit these samples directly to the lab. Please read the atypical myopathy information sheet below to learn more on atypical myopathy and how to collect samples. Payment is required upon receipt of our invoice and can be made via credit card, bank transfer or cheque. Full details are on the invoice. Please complete the owner's plant sample submission form below ensuring contact details are fully filled in. Results are available in 2-3 weeks submission however if you require a more rapid turnaround we offer an expedited service (at a higher cost) and results are, whenever possible, available within 72 hours. These turnaround times are for working days only (Tuesday-Thursday).

• Horse owner factsheet and sample guideline       
• Owner's plant sample atypical myopathy submission form
• Address Label

Prices (including VAT)

• Standard turnaround plant / tree sample test: £148 per sample
• Expedited turnaround plant / tree sample test: £288 per sample

Unfortunately, due to staffing limitations, the Comparative Neuromuscular Diseases Laboratory is unable to discuss individual results or atypical myopathy testing in general by telephone or email. We do provide generic advice on each result sheet about how to interpret your test result and give advice and information about atypical myopathy on our owner information sheet as well as in the frequently asked questions (FAQ) document Please take some time to look through these. We recommend you contact your own vet if you have specific further queries.

Frequently Asked Questions (FAQ) for owners

Muscle biopsy / PSSM1 testing

If your vet has already performed a muscle biopsy or PSSM1 test on your horse, due to RCVS code of conduct and rules on data protection & confidentiality we cannot discuss your horse or the results with you without first speaking with your vet. If you have questions regarding muscle biopsy or PSSM1 testing please ask your vet to contact us directly at neuromuscular@rvc.ac.uk

Genetic testing for polysaccharide storage myopathy in horses

Whilst certain neuromuscular diseases of horses require muscle biopsy for accurate classification, a handful can be diagnosed by genetic testing. This is because these diseases are caused by a specific change in the horse’s DNA, called a mutation, that leads to the specific disease in question. Examples of horse muscle diseases with causative genetic mutations include hyperkalaemic periodic paralysis (HYPP), myotonia congenita, malignant hyperthermia (MH) and type 1 polysaccharide storage myopathy (PSSM1). Whilst mutations can arise spontaneously, often they are passed down from parents to offspring: as such, some mutations and diseases occur more commonly in some breeds, or lines of horses than in others.

Validating a genetic test

Peer review is used when scientists submit a scientific paper for publication; this process occurs when other independent scientists check that an appropriate process has been followed and the science is robust and valid. This is crucial for the advancement of science and veterinary medicine but is especially important when a new test might significantly influence the animal’s future athletic use, breeding potential or a decision for euthanasia.In the case of a genetic test, this validation process would typically involve recruiting a large group of affected horses, that have the disease, and a large group of unaffected horses, that are healthy (and definitely don’t have the disease). Scientists then use statistical tests to see if there is a mutation that is much more likely to be found in affected horses than unaffected horses, rather than simply being there by chance. They would then test a different group of affected and unaffected horses, to see if the same result was found again. Additional experiments can then be used to prove that the specific DNA change (the mutation) has an effect that causes the disease (called functional testing).It is important that the full process of identifying a mutation and proving it causes a disease is peer reviewed and then published for other scientists, vets and owners to read (and if appropriate, critique), so that the test is considered by the scientific community as valid. Basically, this process is fundamental to scientific advancement: it allows us to know if test results can be trusted.

PSSM Type 1 (PSSM1)

Polysaccharide storage myopathy (PSSM) is a genetic muscle disease that causes horses to have episodes of exertional rhabdomyolysis (or tying up) and in some animals causes muscle weakness. PSSM was first diagnosed by muscle biopsy: affected horses accumulate excessive deposits of glycogen (an energy rich storage molecule) in muscle and with time, a substance known as polyglucosan (or polysaccharide) which fills muscle cells [1]. In 2008, a team of scientists in the US identified a genetic mutation in the equine GYS1 gene, and published the results of their scientific validation process proving that the mutation was associated with PSSM [2]. Working in collaboration, we then went on to prove the mechanism by which the mutation causes the disease [3]. Unusually, the mutation increases activity of a key enzyme (glycogen synthase) in muscle that is responsible for storage of glycogen; further, the enzyme (in affected horses), is permanently switched on in muscle cells – so that it continues generating new glycogen even when it is not needed.  However, not every horse with microscopic features of PSSM tests positive for the GYS1 mutation. The ones that do, are referred to as having PSSM Type 1 (PSSM1), whilst the ones that test negative are sometimes referred to as having PSSM Type 2 (PSSM2)[4; 5].Genetic testing for PSSM1 has become very helpful not only for diagnosis, but for breeders making informed and ethical decisions about which horses to breed from. By testing DNA (from a blood sample or hair roots) muscle biopsy can be avoided. Over time, it has turned out that many different breeds of horse carry this same gene mutation across the world, strongly suggesting they share a common ancestor; however, some of these horses display no apparent signs of disease (they are ‘sub-clinical’) whereas others have prominent or intermittently severe signs. Likely, the way these horses are managed plays a significant role in the disorder’s clinical severity but there are probably other aspects that influence the disease (such as breed or fitness). It is important to recognise that some horses with PSSM1 can be very effectively managed and have successful careers. We are working on identifying reasons for differences in clinical severity and specific treatments for this condition.

PSSM Type 2 (PSSM2)

There is currently no scientifically validated genetic test for PSSM2, and scientists still don’t know what causes this disease. Indeed, it remains unclear whether this is a specific disease at all (with a single cause) or whether the term PSSM2 refers to a whole gamut of muscle disorders in horses - some with possible genetic causes but others caused by environmental or other problems. Currently, the optimal way to identify horses as having PSSM2 is via muscle biopsy, albeit with the caveats outlined above. However, because we don’t know what causes PSSM2, or indeed whether it is a specific disease at all, making recommendations for treatments, management or prognosis are speculative at best. Many groups, including our own, are working on these issues. There are companies in Europe and the USA offering a panel of genetic tests commercially for diagnosis of equine muscle diseases including PSSM2. We do not recommend or use these tests for diagnosis of PSSM2 or other myopathies as the results offered have not to our knowledge been scientifically validated in any peer reviewed literature or shared in other form with the scientific community. In short, there is no evidence that the offered tests identify mutations that actually cause muscle disease or indeed, are associated with disease in any way at all. Just like in humans, different horses have millions of differences in their genetic code, but without the scientific validation outlined above, it is impossible to know which (if any) are disease-associated. Furthermore, a group in the US has published two papers [6; 7] testing some of these mutations in different horse breeds that had been examined by muscle biopsy: they found no association between PSSM2 (or any other muscle disease) and the genetic variants offered by these companies. It can be very tempting when your horse has signs that might suggest a muscle problem to seek any answer you can find, particularly when the problem is proving challenging to diagnose and manage. It might be even more tempting to believe an unsubstantiated genetic result when it ‘fits’ with what you have suspected. However, we encourage people to use an evidence basis to decision making when diagnosing these conditions and take into account the information presented above.  Until results of genetic testing for PSSM2 and other myopathies in horses are presented in peer reviewed, validated scientific literature that is accepted by the scientific community (as has been the case for PSSM1), we do not recommend their use and certainly do not support veterinary or life decisions being made based on their results. For help or advice relating to your own horse, please always discuss the issues with your own vet.

References

[1]          Valberg, S.J., Cardinet III, G.H., Carlson, G.P. and DiMauro, S. (1992) Polysaccharide storage myopathy associated with recurrent exertional rhabdomyolysis in horses. Neuromuscular disorders 2, 351-359.  

[2]          McCue, M.E., Valberg, S.J., Miller, M.B., Wade, C., DiMauro, S., Akman, H.O. and Mickelson, J.R. (2008) Glycogen synthase (GYS1) mutation causes a novel skeletal muscle glycogenosis. Genomics 91, 458-466.  

[3]          Maile, C.A., Hingst, J.R., Mahalingan, K.K., O'Reilly, A.O., Cleasby, M.E., Mickelson, J.R., McCue, M.E., Anderson, S.M., Hurley, T.D., Wojtaszewski, J.F.P. and Piercy, R.J. (2017) A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase. Bba-Gen Subjects 1861, 3388-3398.   [4]          Stanley, R.L., McCue, M.E., Valberg, S.J., Mickelson, J.R., Mayhew, I.G., McGowan, C., Hahn, C.N., Patterson-Kane, J.C. and Piercy, R.J. (2009) A glycogen synthase 1 mutation associated with equine polysaccharide storage myopathy and exertional rhabdomyolysis occurs in a variety of UK breeds. Equine Vet J 41, 597-601.  

[5]          McCue, M.E., Armién, A.G., Lucio, M., Mickelson, J.R. and Valberg, S.J. (2009) Comparative Skeletal Muscle Histopathologic and Ultrastructural Features in Two Forms of Polysaccharide Storage Myopathy in Horses. Veterinary Pathology 46, 1281-1291.  

[6]          Valberg, S.J., Finno, C.J., Henry, M.L., Schott, M., Velez‐Irizarry, D., Peng, S., McKenzie, E.C. and Petersen, J.L. (2021) Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis. Equine veterinary journal 53, 690-700.  

[7]          Valberg, S.J., Henry, M.L., Herrick, K.L., Velez‐Irizarry, D., Finno, C.J. and Petersen, J.L. (2022) Absence of myofibrillar myopathy in Quarter Horses with a histopathological diagnosis of type 2 polysaccharide storage myopathy and lack of association with commercial genetic tests. Equine Veterinary Journal. Online Epub    

A downloadable sheet of this information is available here  

Until further notice we will only be open Tuesdays, Wednesdays and Thursdays and can only receive samples on these days. We will be limiting the number of biopsy samples we can receive on each of the three days and these samples will need to be booked in in advance via email please (neuromuscular@rvc.ac.uk) with a minimum of 48 hours’ notice, but preferably with a week’s notice. There may be times, when the service is oversubscribed, that we cannot book your biopsy samples in for the requested day however we will offer you an alternative date or dates. Please give breed, sex, age and brief clinical details when emailing to book in a sample. We will do our best to expedite samples that involve welfare cases but please contact the laboratory to discuss it beforehand. We will only be offering a full panel for equine biopsies at this time, however small animal muscle and nerve sample tests remain unchanged.

For thorough pathological evaluation we require a fresh muscle biopsy that is sent to the laboratory chilled (on ice packs). Keeping the sample cold, but not frozen is crucial to avoid artefact. Although we also suggest that you send a biopsy sample fixed in formalin, this latter sample is only used as a back up, in case of unexpected delays. Many routine tests cannot be performed on formalin-fixed muscle. Use a reliable courier for same day/ overnight delivery.

Please collect and package muscle biopsy samples according to our muscle biopsy procedure and send to the laboratory in a clearly labelled polystyrene box.

We recommend you send in a sample of blood in EDTA (approximately 10ml) and in a plain tube as we bank DNA samples from horses with neuromuscular diseases and in case of further testing. Client and horse details will remain confidential. Blood samples can be sent together with muscle samples in the same package

Equine Muscle Biopsy Evaluation

We will only be offering a full panel at this time. It offers the most detailed evaluation of biopsy samples encompassing routine histological staining which looks at muscle fibre morphology and glycogen content plus special histopathological staining, immunohistochemistry and oxidative stains to assess lipid content, fibre types & mitochondrial enzymes. It is suitable for all cases.

The first muscle is charged at £314.50 (exc. VAT) and additional samples from the same horse submitted at the same time are charged at £123 (exc. VAT) 

Tying up in Warmblood, Connemara and Thoroughbred horses? Discounted muscle biopsy and histology rate offered at RVC

Working together with various partners and collaborators, and with ethical approval, we are embarking on an ambitious programme of work to help elucidate the cause or causes of tying up (or recurrent exertional rhabdomyolysis (RER)) in different horse breeds. Generally, this is a disease that is recognised clinically and then confirmed with muscle biopsy. We are especially interested in tying up / recurrent exertional rhabdomyolysis in Thoroughbred, Warmblood and Connemara horses / ponies – breeds in which this disease is common. If you are a horse owner or vet with a patient within these breed categories that you suspect has RER and for which you or your vet are considering submitting a diagnostic muscle biopsy sample to the Royal Veterinary College’s Comparative Neuromuscular Laboratory, please be aware that we are offering a competitive discounted total fee based on the owner bringing their horse to the RVC’s equine hospital for the biopsy procedure. The total fee (admission, consult, biopsy plus histology report) will be £425. This represents a substantial saving in comparison with what would be normally charged. Unless other investigations are required, generally this can be conducted as an outpatient procedure and the surgery itself will be conducted by our surgery service using a routine, standard open diagnostic biopsy of the semimembranosus muscle in identical fashion to that recommended for vets in practice. By processing the sample immediately following collection we maximise diagnostic quality. If you are interested in using this service, will vets please email the Neuromuscular Laboratory (neuromuscular@rvc.ac.uk) with a brief history and signalment of the horse. We will confirm whether the horse is suitable for this discounted rate and if so, put you in touch with the Equine Referral Hospital to facilitate the referral. Hopefully you get the same excellent service but you can contribute to our work, trying to find the cause of this enigmatic disorder.

Small animal Biopsy Evaluation

In collaboration with Diane Shelton's laboratory at UCSD small animal muscle and nerve samples are evaluated. Serum antibody tests for myasthenia gravis (AChR) and masticatory muscle myositis (2M) can also be run- Collect 1-2mls of serum prior to initiation of corticosteroid or other immunosuppressive therapy. A full list of tests available and their prices (excluding VAT) are available below:

Muscle or Nerve Profile: £347.50

Combined Muscle and Nerve Profile: £392

Additional Muscle or Nerve Profile (sent in at the same time form the same animal): £140.50

2M Antibody test: £133

AChR Antibody test: £133

The required downloadable and printable forms for submitting muscle biopsies from both horses and small animals are available below:

Address label

Biopsy & Blood Submission Form

Muscle Biopsy Procedure For Vets

Facilitation Letter (for use with samples sent from outside the UK from January 2021)

For some practitioners, it may be convenient to send blood or hair plucks(to include follicle) (either separately or with a muscle sample) for genetic testing for the glycogen synthase mutation reported by McCue et al. 2008 seen in many horses with polysaccharide storage myopathy (PSSM / EPSM).

Glycogen synthase (GYS1) mutation causes a novel skeletal muscle glycogenosis. Molly E. McCue, Stephanie J. Valberg, Michael B. Miller, Claire Wade, Salvatore DiMauro, Hasan O. Akman, James R. Mickelson. Genomics 91 (2008) 458–466

Unlike with muscle biopsy samples, there is no restriction on the number of PSSM1 samples we can accept each day however we can only receive samples on Tuesdays, Wednesdays and Thursdays. Samples are run in batches every 2-3 weeks and results are generally available in 1 month from submission. Blood or hair submitted to the laboratory without accompanying muscle samples, may be sent at room temperature via first class post however they must meet royal mail requirements i.e blood tubes should be placed in a secondary container with enough absorbent material to absorb the entire contents of the tube. Please make sure you send samples using the address label and not to the Diagnostic Laboratories at the Hawkshead Campus or addressed purely to the Royal Veterinary College.

5-10mls of EDTA blood or approximately 50 hair follicles are required.

The price for the PSSM test is: £59 (exc VAT)

The address label and submission form for PSSM test are available to download and print below:

Address label

Biopsy & Blood Submission Form

Import Licence (for samples outside the UK)

Genetic Testing for PSSM2

There is currently no scientifically validated genetic test for PSSM2, and scientists still don’t know what causes this disease. Indeed, it remains unclear whether this is a specific disease at all (with a single cause) or whether the term PSSM2 refers to a whole gamut of muscle disorders in horses - some with possible genetic causes but others caused by environmental or other problems. Currently, the optimal way to identify horses as having PSSM2 is via muscle biopsy, albeit with the caveats outlined above. However, because we don’t know what causes PSSM2, or indeed whether it is a specific disease at all, making recommendations for treatments, management or prognosis are speculative at best. Many groups, including our own, are working on these issues. There are companies in Europe and the USA offering a panel of genetic tests commercially for diagnosis of equine muscle diseases including PSSM2. We do not recommend or use these tests for diagnosis of PSSM2 or other myopathies as the results offered have not to our knowledge been scientifically validated in any peer reviewed literature or shared in other form with the scientific community. In short, there is no evidence that the offered tests identify mutations that actually cause muscle disease or indeed, are associated with disease in any way at all. Just like in humans, different horses have millions of differences in their genetic code, but without the scientific validation outlined above, it is impossible to know which (if any) are disease-associated. Furthermore, a group in the US has published two papers [6; 7] testing some of these mutations in different horse breeds that had been examined by muscle biopsy: they found no association between PSSM2 (or any other muscle disease) and the genetic variants offered by these companies.

  [6]          Valberg, S.J., Finno, C.J., Henry, M.L., Schott, M., Velez‐Irizarry, D., Peng, S., McKenzie, E.C. and Petersen, J.L. (2021) Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis. Equine veterinary journal 53, 690-700.

  [7]          Valberg, S.J., Henry, M.L., Herrick, K.L., Velez‐Irizarry, D., Finno, C.J. and Petersen, J.L. (2022) Absence of myofibrillar myopathy in Quarter Horses with a histopathological diagnosis of type 2 polysaccharide storage myopathy and lack of association with commercial genetic tests. Equine Veterinary Journal. Online Epub  

Serum testing for the hypoglycin A toxin and its principal metabolite known to cause atypical myopathy is now available following research funded by The Horse Trust. This is a more rapid test than previously reported methods. We also offer combined profiles including hypoglycin A & acylcarnitine identification and hypoglycin A, acylcarnitine & organic acid identification at special prices. Please send either a whole blood tube, serum gel tube or separated serum using a next day delivery service and use the specific form below. For organic acids profiles please send a urine sample and for acylcarnitines please send a lithium heparin blood tube.

DUE TO THE BANK HOLIDAY EASTER WEEKEND PLEASE DO NOT SEND SAMPLES TO ARRIVE BETWEEN FRIDAY 29TH MARCH AND MONDAY 1ST APRIL. THE LAB IS CLOSED ON BANK HOLIDAYS.

• Vet Submission form for atypical myopathy samples
• Address Label

Whenever possible, samples received before 9am will be reported in 48 hours and samples received after 9am will be reported in 72 hours. Please note these turnaround times are for working days only (the lab is open Monday-Friday)

Prices (all excluding VAT) of the available atypical myopathy tests are as follows:

• Serum hypoglycin A & MCPA- carnitine: £121
• Serum hypoglycin A, MCPA- carnitine & acylcarnitine profile: £229
• Serum hypoglycin A, MCPA- carnitine & urine organic acid profile: £243
• Full atypical myopathy profile (HGA, MCPA-c, organic acids & acylcarnitine profiles): £345.50

Samples from overseas: Samples from abroad can be sent to us. The vet will need to send whole blood (e.g red topped vacutainer tube) or if they have the facility to spin down the blood, just the serum can be sent. The vet will need to send samples on a next day delivery (as sample decomposition can affect accuracy of results) only Monday-Thursday. It must include one of our import licences with the courier paperwork. The sample should be packaged correctly i.e if using a padded envelope the blood tube must be surrounded by enough absorbent material to soak up all of the tube contents and this should all be in a sealed bag. The paperwork should be separate from the bagged tube. 
If the vet takes a sample that cannot be sent immediately e.g. on a Friday, the sample should be spun, the serum placed into a new tube and frozen until ready to send. When sending samples that have been frozen, they do not need to be defrosted before being sent, the package will need to include ice packs and preferably be in an insulated container that will keep the tube cold.

Please follow the protocol for precipitation of samples for lactate pyruvate ratios to ensure samples are correctly processed and complete the specific submission form for this type of sample available below. Samples must be sent to the laboratory on dry ice to avoid thawing using either a same day or next day delivery courier. Please contact the laboratory at least a day in advance to ensure we know when to expect delivery. Samples should not be sent out on a friday as they cannot be processed over the weekend and will be wasted. We recommend that you contact a reliable courier for shipment prior to obtaining the sample. Prices for lactate & pyruvate testing are also listed below.

Pyruvate/Lactate Sample Submission Form

Address label

Prices:

First sample (e.g. Pre-exercise sample): £112

Second sample (e.g. Post-exercise sample): £38.50

Plasma acylcarnitines and urine organic acids are measured in collaboration with Sheffield Children's NHS trust and are often diagnostic in horses with atypical myopathy and other Multiple Acyl CoA Dehydrogenase Deficiencies (MADD). For organic acids please send 10ml of urine in a plain bottle. For acylcarnitines use a lithium heparin tube (not EDTA) for plasma. Please see below for the prices of each test.

Please send these on an ice pack for next day delivery and include a copy of the Biopsy & Blood Submission Form and send to the lab using the address label

If atypical myopathy is suspected please see information above on atypical myopathy testing.

Prices:

Acyl-carnitine Profile: £117

Organic acids Profile: £140

Please contact the laboratory to discuss your requirements.