Position Prof. Valberg zum derzeitigen Stand rund um PSSM/MFM und MIM 

In Richtung equine Myopathien wurde schon Vieles erforscht und die Pferde profitieren endlich von angepasster Fütterung, Haltung und Training. Doch ettliche Zusammenhänge sind immer noch nicht genau bekannt. Nun kam ein neuer Begriff für PSSM2 in Deutschland auf (MIM), man spricht nun auch von MFM bei Pferden und ein Gentest wird angeboten. Wir haben Prof. Stephanie Valberg für Euch um eine Einschätzung dazu gebeten. Hier ihr original Wortlaut vom 11. Mai 2023.  

Foto: andreaskrappweis / Getty Images

Validation of Genetic Testing 

Stephanie Valberg DVM PhD DACVIM, DACVSMR, Emeritus Professor, College of Veterinary Medicine, Michigan State University, Valberg Neuromuscular Diagnostic Laboratory, East Lansing MI, USA 

Carrie Finno DVM PhD DACVIM, Professor, Director Center for Equine Health, University of California-Davis, School of Veterinary Medicine, Davis, CA, USA

Jessica Petersen PhD, Associate Professor, Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE, USA

The goal of our research is to identify new muscle diseases in horses, optimize diagnostic tests, and develop targeted treatments. When validated genetic tests are available, we recommend them over muscle biopsies. Dr. Valberg has been fortunate to be part of the teams that developed genetic tests for PSSM1, GBED and MYHM, thereby eliminating the need for muscle biopsies to diagnose these conditions. When genetic tests are negative, Dr. Valberg runs a diagnostic muscle biopsy service for veterinarians so they can help horse owners identify muscle diseases in their horses. 

It is exciting to see new research into muscle diseases in horses. However, proper validation of newly available genetic tests is essential so they can successfully aid veterinarians with diagnoses, provide owners with answers and peace of mind, and most importantly, benefit the horses that they are intended to help. 

Genetic Tests Should Be Properly Validated Prior to Commercialization 

The usefulness of genetic testing to breeding programs and veterinary evaluations hinges on the accuracy of those tests. If you compare the genome of one horse to that of another, you can uncover millions of genetic variants, many of which are expected to alter protein function.  Finding variants is not the hard part. The challenge is determining which of those variants actually cause the disease.  

Genetic testing in animals is not a regulated industry, so tests for genetic variants can be offered commercially to anyone interested in using them, whether they have been validated or not. Horse owners and veterinarians must decide for themselves if they want to use a particular genetic test. It is therefore essential that accurate information is available to make such decisions. Proper validation of genetic tests is important because inaccurate tests, as well as improper interpretation of test results, can lead to incorrect diagnoses, ineffective treatments, poor breeding decisions, and loss of genetic diversity, among other things.

The international equine genetics community has established standards for the validation of genetic tests and highly recommends following these steps before making a genetic test commercially available.¹ Adhering to the standards for test development outlined below is essential to maintaining veterinarian and owner trust in test results to provide meaningful information to help their horses. 

1 American College of Medical Genetics and Genomics Horse Genome Project, https://horse genomeworkshop.com/values 

Our Investigation of Commercial Test Variants for PSSM2/MFM 

 Following the standards described above, we performed research on the P2, P3, and P4 genetic variants that have been proposed to be associated with PSSM2/MFM. Our results show that these variants are equally common in horses affected with PSSM2/MFM, horses with no evidence of PSSM2/MFM, and horses in the general population, meaning that they are not useful genetic tests for the purpose of diagnosing muscle disease or for breeding management.

Below is a summary of our studies that used the steps required to validate any genetic test (see flow chart). Note that although these diseases have recently been referred to as muscle integrity myopathy (MIM), the veterinary community has not changed the name and will continue to refer to these diseases as PSSM2 or MFM.

 

PSSM2: Horses with signs of tying-up that have abnormal appearing glycogen in muscle cells.

MFM: Warmblood and Arabian horses with signs of reluctance to engage their hindquarters that have abnormal clumping of desmin in muscle cells.

MIM: A non-veterinary term that has recently been used to describe horses that test positive for genetic tests from the Center for Animal Genetics/Equiseq.

1. Identify affected and unaffected (control) animals using the gold standard for diagnosis for PSSM and MFM.

It is important to be as specific as possible in terms of establishing that a disease is present. If a disease has a genetic basis, by including horses with the most severe form of the disease in the data set, you can expect that the vast majority of diseased horses will have the genetic variant and only a very small number of control horses will have the variant if it is responsible for the disease.

The P2 and P3 variants are found in two different genes that each can cause a disease called myofibrillar myopathy (MFM) in humans. The genetic variants P2 and P3 in horses, however, are not in the same place in those genes as the mutations identified in human MFM.  The gene where P4 is located is not known to cause a disease in any species. The initial suspicion of MFM in a human patient is determined based on symptoms of weakness; a definitive diagnosis is made via a muscle biopsy followed by a genetic investigation. In humans, the muscle biopsy shows an abnormal accumulation of a protein called desmin. If P2, P3, and P4 cause MFM in horses, then the most severely affected cases that have high levels of desmin in muscle biopsies (the gold standard for diagnosis of MFM) should have the P2, P3 or P4 genetic variants.

We examined the P2, P3, and P4 variants in 37 Warmblood and 30 Arabian horses with abnormally high levels of desmin as determined through muscle biopsies. The P2, P3 and P4 variants alone or in combination were present in 60% of affected warmbloods and 30% of affected Arabians. In other words, the variants were not consistently present in the most severely affected horses. If these variants were used to diagnose this disease, 40-70% of the cases would be missed.

2. Test for the genetic variant in horses lacking desmin accumulation as well as horses in the general population.

If P2, P3, and P4 are just common variants in the horse’s genome, they will be present at about the same frequency in horses with abnormally high desmin as they are in:

  •  horses with normal levels of desmin (controls)
  • a sample of horses from the general population.

We found that about half of Warmbloods and Arabians that have no evidence of abnormal desmin in their muscle tested positive for a P2, P3 or P4 variant. Additionally, 32% of 209 randomly sampled horses of a variety of breeds tested positive for one or more of these variants. The use of these tests would falsely diagnose MFM in at least 30 - 50% of horses. It is not logical to assume that half of all horses have a disease-causing genetic variant but show no signs of disease. It is much more likely that these P variants are just common genetic variants not associated with disease.

Percentage of horses with P2, P3, or P4 genetic variants*

 

 Warmbloods (37 horses)

 Arabians (30 horses)

 Random breeds (209 horses)

 Affected horses (from step 1)  60%  30%  
 Unaffected horses (from step 2)  47%  50%  
 General population (from step 2)      32%

*The percentage of affected horses with the P2, P3, or P4 genetic variants is not significantly different from the percentage of unaffected horses that also have the variants.

 

Percentage of horses with P2, P3, or P4 genetic variantsTo further evaluate the variants, we examined muscle biopsies from 163 Quarter Horses diagnosed with PSSM2 based on stains for muscle sugars (glycogen) and 188 healthy Quarter Horses (QH) with normal muscle biopsies. Our results showed the variants in almost equal numbers in healthy and affected horses with 60% of both healthy and PSSM2 Quarter Horses having one or more P variants (see figure below).  Importantly, NONE of the biopsies of Quarter Horses had high levels of desmin; therefore, no Quarter Horses had MFM, yet they commonly have a P2, P3 or P4 variant. 

 The conclusion from our research is that the P2, P3, and P4 variants are equally common in horses affected with PSSM or MFM, as determined using the gold standard diagnosis, as they are in the general population of horses from a variety of breeds. Because of this, these genetic variants are not useful for diagnosing muscle disease.

3. Evaluate the effect of the variant on the gene or resulting protein.

It is not sufficient to rely on research in other species focused on variants in the same gene, especially when the variant does not impact the same part of the gene.  The impact of a genetic variant differs based on the species, where it is located in the gene, whether it alters the expression of the gene, changes an amino acid and/or whether it alters the structure or expression of the protein. If the variant causes disease, it will typically alter the gene or the resulting protein’s structure or function. Many “benign” genetic variants exist that do not have an impact on a gene and the associated protein’s function.

To date, no functional data showing the consequences of the P2, P3, or P4 variants on the gene or resulting protein have been demonstrated in the horse. It is important not to identify variants in candidate genes and offer them as diagnostic tests before demonstrating that they actually alter gene and protein function in the horse, resulting in disease.

We are pleased to see that information showing the frequencies of these genetic variants in Welsh cobs is being made public. We encourage the company to publish the steps required to validate a genetic test, as outlined by the international equine genetics community, and described here.

It is important to note that study horses identified by owners do not represent a random sample (e.g., horses could be more related to one another, there could be confounding environmental factors if all horses come from similar areas, trainers, etc.). Owner-reported signs of muscle disease are also not veterinary diagnoses and can overlap with a wide variety of conditions (PSSM1, MYHM, HYPP, gastric ulcers, sacroiliac disease, kissing spine, lameness, saddle fit, training issues, etc.).

If a broad definition of the disease is used (such as lameness and poor performance) then many horses will test positive for the P2, P3 and P4 genetic tests because these variants occur commonly. By chance, some horses that test positive for a variant may actually have a muscle disease and respond to treatment; however, many who test positive will not actually have the disease and will not improve until a proper diagnosis is made. We are happy to see that horses will respond to a change in feeding and exercise regimes, changes in management, however, may benefit a horse regardless of its genotype.


References

  1. Williams ZJ, Velez-Irizarry D, Petersen JL, Ochala J, Finno CJ, Valberg SJ. Candidate gene expression, coding sequence variants and muscle fiber contractile force in Warmblood horses with myofibrillar myopathy. Equine Vet J. 2021 Mar;53(2):306-315. doi: 10.1111/evj.13286. Epub 2020 Jun 25.
  2. Valberg SJ, Finno CJ, Henry ML, Schott M. Petersen JL Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathologic diagnosis. Equine Vet J 2020 https://doi.org/10.1111/evj.13345
  3. Valberg SJ, Finno CJ, Henry ML, Schott M, Velez-Irizarry D, Peng S, McKenzie EC, Petersen JL. Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis. Equine Vet J. 2021 Jul;53(4):690-700. doi: 10.1111/evj.13345. 
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