A report on Drs Meurs & Kittleson works on MyBPC3 mutation gene & DNA-based test.
By Catherine Muller-Sautreau, Ailuropus cattery, France. (Written in 2006, January)
(Reprinted with permission)
A DNA-based test for the myosin-binding protein C gene mutation, which has been proven to be causative for HCM in Maine Coon cats, is now available to breeders.
Disclaimer: I first wrote this paper as a report of what I thought to be Dr Meurs' webseminar highlights, as a Maine Coon breeder who felt concerned about sharing what I had learned there, and then added answers to the frequently asked questions following the lecture. I'm not a geneticist nor a veterinarian.
Dr. Kittleson & Dr. Meurs had the great kindness to review and edit it all, giving more substance to it than I would ever have been able to. Paul Huntley (Lunarcoons Cattery) as well supportively helped me with his comments.
Of course, any remaining mistake or misinterpretation holds on my own responsibility.
Any questions or comments are welcomed at firstname.lastname@example.org.
Previously on "What's up with feline HCM research?"
In the early 1990s when some of the first HCM cases in Maine Coon cats had been reported as a familial issue, Dr. Kittleson from UC Davis built a colony with affected MC cats to study the disease. In 1999, a published study showed the colony's HCM was due to an autosomal dominant trait with complete penetrance but variable expressivity (depending primarily on the sex of the cat).
Last October, Dr. Meurs, Dr. Kittleson and their team published an article that identified a genetic mutation that causes HCM in every affected cat in the colony's family of cat. That mutation has been isolated by genetic sequencing. This is the genetic mutation the DNA-based test looks for.
The identified gene codes for the cardiac myosin-binding protein C (MyBPC). The mutation was found in the very beginning of the MyBPC3 gene. Don't worry, you're going to get used to using these words soon!
On 2005, December 21st, Dr Meurs has given a two-hour lecture broadcast through the Web, entitled "Inherited hypertrophic cardiomyopathy in the cat".
Dr. Meurs' lecture's report: understanding what this DNA test does test for
The lecture began with a reminder about what HCM consists in:
- This is the most common heart disease in cats. All HCM forms are not one and the same (different clinical signs; different severities; different appearances on an echocardiogram).
- The ventricular walls thicken (on the left ventricular side).
- Consequently, the heart on a whole has a bigger size.
- All those symptoms may induce potential secondary symptoms:
- Heart failure
- Systemic embolism (most commonly a blood clot to the large blood vessel that supplies the back legs causing acute pain and paralysis)
- Sudden death
- Onset age, disease evolution and disease seriousness vary a lot from one individual to another, including within the same family or line.
The MyBPC3 mutation
A point mutation (one nucleotide is changed), on one of the genes that causes HCM in humans, has been identified as causing HCM in Maine Coon cats through sequencing the gene.
This mutated gene is:
- Dominant. There's no "healthy carrier". One defective allele out of the pair (heterozygous state) is enough for it to induce the disease. It is dominant because it codes for a structural protein in the heart. Recessive genes more commonly code for proteins like enzymes where almost all of the protein must be abnormal to produce disease. A dominant disease makes 50% of the protein abnormal by affecting one allele (mammals have two alleles that produce a chromosome).
- Autosomal. It means both genders can carry it and develop HCM because of it.
- With variable expressivity. The severity of the disease and the onset age vary from one individual to another, even within the same family (or line).
- With complete penetrance in adulthood. Even if expression varies, all individuals are likely to develop HCM because of this mutation at some point in their adulthood.
This mutation concerns a gene that codes for cardiac myosin-binding protein C (MyBPC). Mutations on this gene are one of the most common cause of HCM in humans.
This protein is one of the cardiac sarcomeric (or contractile element) proteins. The cardiac sarcomere is the contractile unit in each cardiac cell. It is made of "sliding" proteins. Thanks to this "sliding" ability, the heart can contract.
On the schema below, the myosin-binding proteins C consist in the blue "bubbles". (These proteins attach to several proteins in the sarcomere to maintain stability. The actin proteins are the red bubbles and the myosin proteins are the green-greyish filaments: they "slide". The myosin heads are the big yellow clumps that have to ratchet and attach to the actin's fixative points in order to produce movement. That's how the cardiac cells normally "shorten", and hence the cardiac muscle actually tenses).
More precisely, this mutation concerns one specific codon (a codon is 3 nucleotides - DNA or RNA "units" - that tell the genetic machinery within a cell to produce a certain amino acid) in the cardiac myosin binding protein C gene sequence: instead of a GCC codon (guanine-cytosine-cytosine), there's a CCC codon.
Consequently, when the cardiac myosin-binding protein C is built in the cell, the CCC codon induces the formation of a proline amino acid in the protein instead of alanine. Hence the protein built is defective.
The defective protein conforms abnormally theoretically disorganizing half of the cardiac sarcomeres and their ability to contract is abnormal. As a compensatory phenomenon, the heart muscle produces new sarcomeres to compensate and each time it does this it has a 50 : 50 chance of producing a normal sarcomere, which results in the muscle thickening. The thickening is called hypertrophy.
Practical considerations about the DNA-based test
The test identifies, in the DNA sequence for the MyBPC allelic pair, whether there're GCC codons (regular allele) or CCC codons (mutated allele).
Practically speaking, this means a positive test is fully reliable: a cat who tests positive will probably develop HCM at some time in its life and pass on the mutation to its offspring (half of it if heterozygous; on to its whole offspring if homozygous).
- A positive test doesn't indicate when and to what extent the cat will develop HCM. Those variations are most likely induced by polygenic interactions, many of which are still unidentified. The one identified so far is sex - males get the disease at a younger age and usually have a more severe form of the disease.
- It doesn't screen for any other mutations on that gene or on any other gene that codes for a cardiac protein. For instance, in humans, there are over 200 mutations in genes for 10 different cardiac sarcomeric proteins, which all induce an inherited HCM (as an autosomal dominant trait also). Practically speaking, this means a negative DNA test may not guarantee the cat won't develop HCM, because there may be another causative mutation.
What this test does test for: it eliminates one inherited cause of HCM in a Maine Coon cat if the test is negative. If the test is positive, it ascertains the cat will likely develop (or has) HCM and it can do so before there is any evidence of HCM on an echocardiogram.
What this test doesn't test for: it doesn't test for all potential causes of inherited HCM. At this stage it is unknown if this mutation causes 99% of the HCM in Maine Coon cats or 10%. Consequently, it is still necessary to keep having echocardiograms performed by a competent cardiologist.
Something to know about: This mutation has been found in a domestic cat as well. On the other hand, at least two Maine Coon cats who had been echoed and diagnosed with HCM were negative for this specific mutation.
Practically speaking, this means there's probably at least one other cause for HCM in our breed.
At the beginning of the Webinar, Dr. Meurs had an essential message passed on: as far as biology is concerned, especially when molecular biology is concerned, there're no black and white answers. This test identifies one cause of HCM in Maine Coon and possibly other cats; this is one step we can use to deal with the disease and if this mutation is prevalent enough, to deal effectively with the disease in our breed.
Right now, there're no population statistics available. The basic reason for this is the DNA test hasn't been performed on a large enough number of cats to make scientific inferences out of it.
How can I have my cats tested for this mutation?
Here's the link to the providing test lab's webpage.
To ask for DNA sampling kits click here.
You'll get the sampling kits through mail.
To collect cheek swab samples:
Have your veterinarian do it if you're not sure you can handle it.
- Don't let your cats eat for at least one or two hours before you collect the samples (otherwise you will collect tuna or chicken DNA!).
- Carefully wash your hands (otherwise you will collect human DNA!). Have somebody else, whose hands have been carefully washed too, help you firmly hold the cat's body and head. Don't touch the swab!!!
- Open the cat's mouth and introduce the brush between its cheek and gum.
- Those are cells, not saliva (saliva doesn't even contain cells), you're collecting. So brush your cat's gum in a gentle but firm enough way, turning the brush several times to make sure it's full of cells. Epithelium must be seen on the brush when you're done (basically, it looks like tiny pieces of fish meat).
- Use two brushes per cat (this is a security thing).
- Don't let the blow-on-things-to-make-them-dry reflex overwhelm you!
- If it's possible, let the samples dry for a few hours, without touching them, and then put them back into the blister.
- Carefully wash your hands between collecting samples on different cats.
The test costs 60 $ per cat, and 51 $ per cat if you test 5 cats or more. You can pay by international money order. Visa/mastercard payment is available as well.
Some frequently asked questions
This is complicated material, but totally rational as well. If we understand the facts, there will be less misinformation spreading over the internet and passing on from one breeder to another. If you feel uncomfortable with genetics or biology as a whole, you can always ask for help from someone who isn't and get advice about reliable books or websites to start with. This is a worthy investment.
Is this test reliable?
This test is 100% reliable (OK: 99,999%) for the very question it is asking: does this cat have or does this cat not have this specific MyBPC mutation in its genotype (DNA)?
There are no "false positives": a positive test means the cat has the mutation and should not be bred. It is also likely to have or develop HCM, no matter if it shows signs of the disease or not. There are no "healthy carriers". As this mutation is a dominant one, it doesn't matter whether the cat is heterozygous or homozygous. To put in very plain terms: if the cat has the mutation, the cat is HCM positive.
The DNA test being used (sequencing) is reliable. There's no magic about it: these are specific DNA molecules being sequenced. A mutated codon (CCC) can't be mixed up with a "regular" one (GCC). No tricky false positives!
There are no false negatives either. The two Maine Coon cats with HCM whose DNA test was negative are not "false negatives". The negative result doesn't mean the test doesn't work, it means the cat has developed HCM for another reason (most likely a different mutation) than this causative mutation. It doesn't change the fact this mutation is causative, and a cat diagnosed for HCM via an echocardiogram still has HCM.
Do yourself a favour: never use such phrases as "false positives" or "false negatives" when dealing with DNA-based tests. They're uselessly messing up with people's heads.
What about other genetic factors that may determine when the disease becomes evident or how severe the disease becomes?
If we stay facts-oriented: this mutation has been proven to cause HCM in the Maine Coon. This is not controversial data. This is a primary cause of HCM. Schematically speaking, a "primary mutation" means its sole presence is enough for it to cause the disease.
So it doesn't really matter if other genetic factors or interactions determine when the disease becomes evident or how severe it may become. The test does provide a totally reliable answer when the outcome is positive - the cat has the mutation and will pass it on to its offspring.
Other genetic factors may be important to an individual cat's health but is not important when it comes to breeding decisions.
Why do we need to keep having echocardiograms done on our cats?
Since there are over 200 mutations identified in humans with HCM we already were pretty sure that different breeds would have different mutations and that there could be more than one mutation in Maine Coons. Now preliminary results suggest that there is more than one mutation causing HCM in Maine Coon cats.
A negative test means this cat is negative for this specific mutation and won't develop HCM because of it. But it doesn't guarantee he won't develop HCM from a different mutation. That's why an echocardiogram is still required, since echocardiography doesn't look for the cause, but for the whole effect.
Go and have the screening performed by a trained cardiologist. Have at least a 2D, M-mode and color Doppler screening performed. Ask for the measurements. If you can get a Doppler tissue imaging screening, never hesitate to.
Echocardiograms still have to be performed at the same frequency as recommended before, especially in this early stage where we do not know the prevalence of this mutation in our breed.
So if I still need to have my cats Doppler-screened at the exact same frequency, what has exactly changed?
With a negative MyBPC mutation-test you get rid of one potential cause for HCM. That's sounds like good news enough!
With a positive MyBPC mutation-test you know the cat will pass on the mutation to its offspring (remember there are no "silent carriers" as in an autosomal recessive trait). The important thing is you know it before you make a breeding decision. This alone makes the test worth doing.
- When an heterozygous cat is mated with an unaffected cat, mutation will be theoretically passed on to 50% of the offspring.
- When an heterozygous is mated with another heterozygous, mutation will be theoretically passed on to 75% of the offspring.
- When an homozygous is mated to another cat, no matter whether it's homozygous, heterozygous or unaffected, mutation is necessarily passed on to 100% of the offspring.
There's no healthy mating scheme with such a dominant trait, no matter whether the cat is heterozygous or homozygous.
Can we get certified results?
On breeders' request, Dr. Meurs has added an optional microchip ID # on the information form to fill in. So you can have the samples collected by your veterinarian and have him check the microchip (it can be useful too to go at your veterinarian's if you're not sure how to collect a cheek swab sample). But the form is for the lab's use, so you won't get it back.
Dr Meurs has declined to participate in any breeder's results-collecting program. As a researcher, it's not her role. Collecting data, storing it, and deciding how to make it available to other breeders is up to our breed organizations. This will be a big challenge to many individuals to get this done and will be a challenge for all of us to provide all the data we collect and do it in an honest way.
Why shouldn't we wait until we know more?
Here're Dr. Kittleson's words: As for samples, the Maine Coon breed at this stage needs all of the samples it can get, especially from affected cats, to see how prevalent this mutation is in the breed. So there is no funded study currently going on in the strict sense of the term. Data are obviously being generated by Dr. Meurs lab as she runs samples but each sample has to generate its own money (has to be paid for) as there is no grant for this. Until a large sample is looked at from numerous pedigrees (and countries) no one knows what sort of prevalence there is and that's obviously critical to the breed. So I would say that anyone that is holding back from testing at this stage is doing a huge disservice to the breed they love. So my message is clear - if you have a Maine Coon cat with HCM it and its relatives need to be tested as soon as possible. Otherwise the mutation is being passed on to innocent cats as individuals wait to see what happens.
For extra reading on HCM, you can visit Jody Chinitz's website.
 Kittleson, M.D., et alii, Familial hypertrophic cardiomyopathy in maine coon cats : an animal model of human disease, Circulation, 1999 Jun 22; 99(24): 3172-3180.
Full text can be read at: http://circ.ahajournals.org/cgi/content/full/99/24/3172
 Dr. Kittleson's additional piece of information.
 Meurs, K. M., et alii, A cardiac myosin binding protein C mutation in the Maine Coon cat with familial hypertrophic cardiomyopathy, Hum. Mol. Gen., 2005 Dec 1; 14(23):3587-93. Epub. 2005 Oct 19.
Abstract can be read and full article can be purchased at: http://hmg.oxfordjournals.org/cgi/content/abstract/14/23/3587
 Dr. Kittleson's additional piece of information.
 Kittleson et alii, Circul., 1999, 3178: HCM in these cats appears to be inherited as an autosomal dominant trait, as it is in human FCHM, with penetrance that increases to 100% in adulthood. Although penetrance may not be 100% in some human families with FHCM, often it is complete, as in our cats.
 If you want to learn about proteins synthesis, you can visit this page and watch the "Translation" and "The three steps of transcriptions" animations.
 Dr. Kittleson's additional piece of information.
 Here are some instances of useful links :