1. WHAT IS EQUINE MYOFIBRILLAR MYOPATHY (MFM)?

Terminology: Myofibrillar myopathy or MFM is a term used to describe a new disease our research group has identified in horses, particularly Arabian and Warmblood horses. Previously some horses with MFM have been diagnosed with type 2 PSSM because the breaks in myofibrils created gaps that were filled in by muscle glycogen. It is important to note that not all horses previously diagnosed with type 2 PSSM have MFM.

The name myofibrillar myopathy comes from a description of the physical changes we have identified in the muscle cells of affected horses. These changes involve disruption of the orderly alignment of the contractile proteins called myofibrils.

Myofibrils
A normal muscle cell that contains an orderly alignment of contractile proteins arranged within myofibrils that are packaged longitudinally the muscle cell
Extracellular Matrix
A normal portion of a muscle cell on the left with a normal amount of a protein called desmin that maintains the orderly alignment of myofibrils. With a myofibrillar myopathy, breaks in the myofibrils occur (right image) and clumps of desmin form near breaks. Source J Clin Invest. 2009;119(7):1806-1813.

A diagnosis of MFM requires a special stain for desmin, there is no validated genetic test for MFM. Equine MFM does not necessarily have the same underlying cause or the same clinical severity as human disorders that are called myofibrillar myopathy. Do not expect to find the same symptoms in horses as you see described for humans. Human and equine myofibrillar myopathy share the same name because they have a similar but less severe appearance under the microscope, in the horse’s case they often milder symptoms than humans.

2. IS THERE A GENETIC TEST FOR EQUINE MFM?

There are no scientifically validated tests for type 2 PSSM, recurrent exertional rhabdomyolysis (RER) or myofibrillar myopathy (MFM) as of April 2018. In our professional opinions, decisions about treating, selling, breeding or euthanizing horses should not be made based on a genetic test that has not been scientifically validated.

Genetic testing in horses is not regulated in the US, therefore it is up to animal owners and veterinarians to determine if a genetic test does in fact identify susceptibility to a clinical disease or a specific trait. In other words, users of the test need to critically question if the existence of the genetic variant being tested has been shown to truly indicate the presence or susceptibility to a disease. This determination should be informed by asking genetic testing laboratories for their peer reviewed publications, looking for publications on websites like Google Scholar and by consulting veterinarians and experts in the field.

Peer reviewed scientific publications are the traditional means by which a genetic test is validated to provide concrete evidence of the involvement of a genetic variant in a specific trait. (See the section below on steps required to validate a genetic test). In the age of social media, some genetic tests are being popularized on Facebook or other media outlets in the absence of validation and verification by scientific publication and peer review.

Genetic testing for PSSM2

Dr. Valberg is not currently using or recommending the use of genetic testing for the diagnosis of type 2 PSSM or MFM as there is currently no scientifically validated evidence that the variants for which genetic testing is available are linked to PSSM2 or MFM. When comparing the genome of one horse to another, there are as many as 17 million genetic variants that make an individual unique. In other words, it is common to find genetic variants between horses, even in important and highly functional genes. It is also common to find genetic variants that are shared between horses. The difficulty with genetic disease research is not in finding variants, but rather is determining whether a specific variant is responsible for a specific disease.

The current P2, P3 and P4 genetic tests have recently been identified on social media sites as involving variants in the genes myotilin, filamin C and myozenin 3. To date, the actual LOCATION of the P2, P3 and P4 variants within those genes (i.e. chromosome # and base pair location) have not been identified. Identifying the genes does not inform other researchers of the exact DNA sequence that is altered. As an example, if the entire box shown below is a gene, each letter would represent part of the genetic sequence. The blue T represents a variant.

Dna Sequence

When a mutation is identified, it is not just the box that is identified, it is typically the LOCATION within the gene (i.e. the specific DNA sequence [blue “T”]) that would be identified in addition to the name of the gene. Information on the location of variants being tested as P2, P3, P4 has not been provided.

Researchers from Michigan State University, University of California-Davis and University of Nebraska-Lincoln have analyzed the entire genetic sequence of PSSM2/MFM horses and horses with no indication of muscle disease. The horses with PSSM2/MFM were carefully diagnosed using clinical history, physical examination and muscle biopsy. Our research found over 30 genetic variants (synonymous and nonsynonymous) lying within the named genes myotilin (P2), filamin C (P3) and myozenin 3 (P4). None of these variants were consistently present in horses diagnosed with PSSM2/MFM and consistently absent or at low frequency in healthy horses. The variants are found across a wide range of breeds, whereas MFM is found in a limited number of breeds. In other words, this research supports a conclusion that variants in the 3 named genes are not necessarily associated with the disease. Our results are also consistent with anecdotal reports on social media of genetic test results from horse owners that state that at least one of these P variants is present in a high number of horses not displaying clinical signs of the disease. If a variant is present in a high number of horses with no signs of disease it is not a clinically useful test.

What is a scientifically validated genetic test?

When veterinary genetic testing has been scientifically validated it means the following steps have been taken:

  1. A diagnosis of disease has been carefully established in a group of “affected” horses and confirmed to be absent in a group of “unaffected” (i.e. non-diseased) horses. This diagnosis should be based on the highest standard for testing for a particular disease (i.e. blood test, tissue biopsy, etc.)
  2. A change in the genetic sequence (called a genetic variant) has been identified in the diseased horses that passes statistical tests showing it is significantly associated with the presence of the disease.
  3. The test of association between the variant and the disease is replicated in additional, separate populations of diseased and healthy horses to ensure the accuracy of the association. The frequency of the variant across breeds is reported.
  4. The genetic variant is examined to establish that it changes the function or regulation of a protein or at the very least carefully modeled to show how it alters molecular biology to create the specific disease.
  5. Most importantly, as part of the publication process, a careful peer review is conducted by scientists. Peer-review allows the results to be evaluated by other scientists who examine the methods used, results produced, and conclusions reached by the investigators. If accepted for publication, the article identifies and describes the genetic mutation (location of the sequence change and gene involved). Publication also allows others to attempt to replicate the findings. True, disease-causing variants will stand up to this scrutiny.

To see an example of how scientific validation works see the recent open access publication on the genetic basis for immune-mediated myositis (IMM) in horses (https://www.ncbi.nlm.nih.gov/pubmed/29510741).

AQHA 5 panel test

For perspective, all of the genetic tests in the AQHA 5 panel test have passed the five steps necessary for scientific validation and were not offered to the public until this validation and verification was complete. The mutations in the specific genes for these conditions have been published in peer reviewed journals - meaning other scientists have reviewed the evidence and agree with the findings. Importantly, the genetic results are reproducible by other researchers verifying the usefulness of the tests in breeding or disease diagnosis. The proportion of horses with the disease and genetic mutation as well as the proportion of healthy horses with the genetic mutation is also known and published. For example, approximately 80% of Quarter Horses with PSSM as identified in a muscle biopsy were shown to have the GYS1 mutation causing PSSM1; that mutation was shown to be absent in 90% of random healthy Quarter Horses. This is the type of specific finding which verifies the usefulness of genetic testing.

Conclusion

Peer review and publication is the foundation of sound scientific research. Marketing tests as providing conclusive answers to complex genetic diseases before scientific peer review may result in misdiagnosis with adverse consequences.

For more information about the pitfalls of poorly validated genetic testing see the article in the prestigious journal Nature entitled “Pet genomics medicine runs wild”.

https://www.nature.com/articles/%20d41586-018-05771-0

3. HOW IS A DIAGNOSIS OF EQUINE MFM ESTABLISHED?

A diagnosis of MFM is made from a muscle biopsy. In some cases PAS stains for glycogen reveal aggregates of amylase-sensitive or resistant PAS positive material, likely glycogen, clumped in the cytoplasm of the muscle fiber and under the cell membrane. This does not occur in all cases of MFM. Pooling of glycogen between the breaks in myofibrils is the reason why many cases of MFM were previously diagnosed with type 2 polysaccharide storage myopathy. Once special stains were developed to identify MFM, we recognized this to be a separate disease that can have overlapping features of type 2 PSSM under the microscope. MFM is diagnosed based on identifying aggregates of a cytoskeletal protein called desmin in immunohistochemical stains. We have found that horses with MFM will have scattered muscles cells that develop clumping of the cytoskeletal protein desmin. Normally desmin is found at one specific repeating place in the myofibril, the Z-disc, and it acts to hold myofibrils in proper alignment across the muscle cell. In horses with MFM, sections of a muscle cell start to produce abnormal amounts and shapes of desmin, likely as a reaction to instability in the contractile proteins.

A false positive diagnosis of MFM can occur if the muscle biopsy is taken from a horse with actively regenerating muscle fibers, but an experienced evaluator can see that these fibers are regenerating. A false negative diagnosis can occur if a horse is too young, if muscle samples are too small or if muscle biopsies are not kept chilled and shipped quickly to the laboratory. Immunohistochemical stains are quite sensitive to degradation. Therefore, we ask that each biopsy be split and one section placed in formalin for the desmin stain. Abnormal desmin staining may not be obvious in horses that are less than 8 yrs of age. It is possible that younger horses have MFM but at this point desmin may not yet have accumulated in enough muscle fibers to identify the disease.

Desmin Stain Mfm
Normal Desmin Stain Muscle

A desmin stain of skeletal muscle from a horse with MFM is shown on the left. Abnormal red clumping of desmin occurs in scattered muscle fibers of horses with MFM. A normal desmin stain of equine skeletal muscle is shown on the right where a normal amount of desmin is seen under the cell membranes.

4. WHAT BREEDS ARE DIAGNOSED WITH MFM?

The majority of horses we have diagnosed with MFM have been Arabian and Warmblood horses or their crosses. A few Thoroughbreds, Quarter Horses and Paso horses have also been diagnosed with MFM by muscle biopsy. The research is in very early stages so we do not have an estimate of how common MFM is in equine breeds.

5. WHAT CAUSES MFM?

The cause of MFM in horses is unknown. There is not yet definitive proof this is a genetic disease, but we suspect there may be a heritable component to the disease. We are currently performing genome-wide association studies, analyzing gene expression and sequencing the genome of horses with MFM. If you have families of horses you suspect have MFM and would be willing to have us examine them please contact us, we would be interested to hear from you.

6. WHAT ARE THE SIGNS OF MFM IN HORSES?

In Arabians, particularly those competing in endurance, the most common clinical sign is intermittent tying up (episodes of muscle pain, stiffness, reluctance to move). The severity of muscle stiffness can be much milder than that seen with classic tying-up (exertional rhabdomyolysis). After an endurance ride horses may just appear slightly stiff and have dark urine without being completely unwilling to move. If horses are rested for a few weeks, marked muscle stiffness often occurs approximately 5 miles into a light ride. Increased serum CK and AST activities can occur but may not consistently be as high as in classic forms of tying up.

Warmblood horses with MFM may have a history of satisfactory performance as young animals but a decline in performance as they reach 8 -10 yrs of age. Tying up is usually very intermittent or absent in Warmbloods. The most common clinical signs of MFM in Warmbloods are related to poor performance without elevations in serum CK and AST activity. Unwillingness to go forward, a vague, poorly localized hindlimb lameness, sore muscles and drop in energy level are common complaints. Reluctance to collect and engage the hindquarters, poor rounding over fences, poor quality of the canter and slow onset of atrophy especially when out of work are reported. Note that these are not specific signs to MFM and they can occur with poor saddle fit, lameness from hocks, stifles, suspensory injuries, sacroiliac disorders, inflammation in the thoracic or lumbar vertebrae and more. A thorough evaluation for lameness to rule out these more common issues should precede any testing for MFM in horses because MFM is likely much less common than these other equine maladies.

7. HOW DO I MANAGE A HORSE WITH MFM?

First, obtain a specific diagnosis so you know what you are dealing with. If horses have signs of exercise intolerance, we strongly recommend having a complete veterinary examination. There are many forms of lameness that can cause clinical signs similar to MFM and a thorough evaluation is needed before a muscle biopsy and changes to diet and exercise regimes.

Signs of muscle pain, atrophy and stiffness in horses with MFM can be managed in many horses through specific diet and training regimes. Some horses, unfortunately do not respond to these changes or only partially respond and are not capable of the level of dressage or jumping they were initially envisioned to achieve. Very importantly, diet alone cannot cure MFM and a gradual incremental training program must be instituted to potentially see a beneficial effect. Consistent exercise with lots of stretching long and low warm up is important for MFM horses.

Avoid Rest: For chronic cases, prolonged rest appears to be counterproductive. For many horses that are happy with being outdoors, daily turn out with compatible companions can be very beneficial. Some horses appear particularly sensitive to fresh grass and grazing muzzles may be of benefit when grass is lush. If stress is a precipitating factor, stressful events should be minimized.

Reintroducing exercise: Re-introduction of exercise after several days of rest or an episode of stiffness needs to be gradual. Exercise should begin with work on a longe-line or under saddle beginning with once a day for 3-5 minutes at a walk and trot. This initial work should be very mild to begin with including a walk and trot and very short in duration (5 min max). Very gradually increase the amount of work each day. At least two weeks of walk and trot for 2-3 weeks should precede work at a canter if horses that have been out of work. Core strengthening exercise described by Dr. Hilary Clayton and Narelle Stubbs are recommended. These can be found in their book “Activate your horse’s core”. https://www.amazon.com/Activate-Your-HOrses-Core-Unmounted/dp/0974767018

Horse

Exercise: A proper warm up that engages topline and core muscles is very important for horses with MFM. Horses should be worked in a long and low frame for 5 – 15 min a day. The frame is very important. Vienna reins, a neck stretcher or a Pessoa-like system are recommended over side reins to get a long and low stretch. Relaxation at the base of the neck, stretch over the topline and engagement of abdominal muscles are important during longing warm up. As the horse warms up, more tracking up and hind end engagement can be asked for. When riding, collection should be preceded by this warm up, introduced gradually and performed in intervals. Hill work, poles and cavaletti can all be introduced gradually to strengthen core muscles. Regular daily exercise is extremely important for managing horses with MFM.

Dressage Setup
A great set up to strengthen a dressage horse’s core strength.

Diet: Exercise seems to be much more important than diet for MFM horses. The details of the best diet for MFM remain to be worked out. The principles we are working from are designed to increase turnover of structural proteins in the muscle and build muscle strength. At present we recommend a diet with low to moderate starch and sugar content, fat added if needed for weight and the addition of a whey-based protein supplement to add muscle bulk. We strongly encourage owners to utilize an equine nutritionist to provide a balanced diet. Most feed companies have a nutritionist that will provide support for veterinarians or an independent nutritionist can be consulted. Progressive’s topline Xtreme or Purina’s Supersport are two whey based amino acid supplements we have tried. Feeding protein within 45 min of exercise may enhance development of muscle mass. If horses have evidence of a cresty neck, excessive fat pads or a history of metabolic syndrome or laminitis, sore feet or kidney disease consult your veterinarian before feeding higher protein feeds.

Expectations: At this early stage, we don’t have a full understanding of how horses respond to diet and exercise or the scope of this disease. There are several top level endurance horses with this condition that are being managed by their owners to reduce episodes of tying-up. Other Arabian endurance horses have been retired and used now as trail horses. For Warmbloods, some horses have been consistently and gradually worked on a daily basis and have achieved upper levels of dressage. In most cases to date, however, horses have not been able to achieve the level of dressage or 1 meter jumping performance originally envisioned by their owners. Many of these horses are able to perform as lower level hunter and jumpers. A few Warmblood horses appear to be severely afflicted and have been retired because of extreme exercise intolerance.

8. WHERE CAN I FIND OUT MORE ABOUT MFM?

Please do NOT use the human literature as a guide to understanding equine MFM, the clinical picture IN HUMANS does not corelate well with horses.

Current research publications include:

Valberg SJ, McKenzie EC, Eyrich LV, Shivers J, Barnes N, Finno CJ. Suspected myofibrillar myopathy in Arabian horses with a history of exertional rhabdomyolysis. Equine Vet J. 2016:48;548–556.

McKenzie EC, Eyricha ME, Valberg SJ. Clinical, histopathological and metabolic responses following exercise in Arabian Horses with a history of exertional rhabdomyolysis. Vet J. 2016 Oct;216:196-201.

Valberg SJ, Nicholson AM, Lewis SS, Reardon RA, C.J. Finno. Clinical and histopathological features of myofibrillar myopathy in Warmblood horses. Equine Vet J. 2017 Nov;49(6):739-745.