PHPT

Primary Hyperparathyroidism: DNA Making Inherited Disease History

 

In early August of 2006 The Third International conference on “Advances in Canine and Feline Genomics and Inherited Diseases” was held at the School of Veterinary Medicine, University of California – Davis.  The delegates read like a “who’s who” of the leading genetics experts from around the world. Dr. Richard Goldstein presented a paper on “Genetic analysis and mapping of primary hyperparathyroidism in Keeshonden.” He announced that research into the genetic cause of primary hyperparathyroid disease in the Keeshond had been successful and the locus of the gene responsible for the condition found. He confirmed that a DNA test to show the status of any Keeshond would be available within a short period of time.

 
Anni far left with her litter sister Lucy, niece Lily and great niece Kaja on her last holiday on Exmoor – Sept 2001.

This was the long awaited answer to a disease that had been known in the breed since the 1980s. (Berger B, Feldman EC – 1987) It had been 4 ½ years since my home bred bitch Anni (Liefkees Anneliese) died as a result of renal failure despite successful surgery the previous year for a parathyroid tumour and the search for an answer began. ‘Anni’s Story’ was written so that other owners could be made aware of this disease. Read ‘Anni’s Story’.

After Anni’s death my vet Sue received a call from the consultant at Cambridge asking if I could send her the pedigree and offering to research the condition in the breed. The condition was already well known in the USA and Anni had American bloodlines. The project was handed over to Dr. Barbara Skelly of the Department of Veterinary Medicine at the University of Cambridge and we have worked closely ever since.

What is primary hyperparathyroid disease?

Canine primary hyperparathyroidism is a late onset disease which causes a dog’s blood calcium to abnormally increase. The condition is considered to be rare in dogs and most cases are sporadic, do not run in families and may just represent spontaneous change in the parathyroid glands. Research in the USA as long ago as the 1980’s has shown that the Keeshond has a predisposition. A number of papers in recent years have cited that the Keeshond is over represented in referral practices both in the USA and the UK.

The four rice sized parathyroid glands are situated on each side of the thyroid gland and are responsible for secreting a hormone that regulates the calcium phosphorous ratio in the body. The parathyroid glands are not functionally related to the thyroid, merely adjacent to them (para meaning beside). A condition known as primary hyperparathyroidism occurs when small, usually benign tumours grow on one or more of the glands. This in turn causes them to become overactive and secrete too much parathyroid hormone (PTH, parathormone) into the bloodstream. The result of this action is that the calcium levels in the blood rise and hypercalcaemia results.

Undetected this situation will cause calcium to be drawn from the bones and in extreme cases can lead to spontaneous fractures. Excess calcium is also laid down in the kidneys. The unaffected parathyroid glands become suppressed in an attempt to normalise the calcium levels. If undetected irreversible damage is done to the kidneys and other major organs, leading to death.

The main problem with the disease is that the symptoms shown can be so easily explained by the ageing process. These include polydipsia (increased water consumption) and polyuria (increased urination), an increasing stiffness of gait, lethargy, inappetance, exercise intolerance, vomiting, weakness, eventually the affected dog dies of what seems like old age, except it isn’t necessarily that old. The disease generally has a late onset, 7 years plus (average age 10 years) and dogs can die as young as 8 or 9 years, usually of kidney failure.

When once owners are aware of the risk to their older kees, then monitoring can be done via an annual calcium blood test for dogs of 6 years and over. If a dog has raised calcium levels (normal range 1.98 – 3.00 mmol/l) a further test of parathormone levels will confirm diagnosis of PHPT This test is only available at specialist laboratories.

The surgery itself is relatively non invasive but does need to be performed by a specialist surgeon as the glands are so small and easy to miss. The crucial part of the treatment is the aftercare as dogs need to be carefully monitored for 5 – 7 days post surgery as there is usually a sharp drop in calcium at this stage. When once the offending gland has been removed the others need to be coaxed back into action and supplementation with vitamin D and calcium are used to support the dog until the other glands normalise and begin normal parathormone production. Vitamin D helps the body to absorb available calcium from the gut and the calcium ensures that the dietary intake is adequate.

Prognosis is excellent as long as kidney damage has not occurred, however without treatment, dogs with PHPT may eventually die of complications caused by the increased serum calcium.

DNA research commences

When once it was realised that there was a definite pattern of inheritance Dr. Barbara Skelly applied to the Kennel Club Charitable Trust (KCCT) for funding to investigate the gene causing PHPT. The first funding award of £10,000 was awarded in early 2003 and Dr. Skelly set about collecting blood samples from Keeshonds. She soon had over 100 samples and by offering free testing of calcium began identifying dogs with hyperparathyroid disease. A number of dogs were treated as a result and have made good recoveries. As dogs were identified with the disease Barbara started contacting owners of close relatives and in doing so was able to track the spread of the disease onto a large genetic map (extended family tree). The mode of inheritance behaved like an autosomal dominant trait but this needed to be proven.

The starting point for research was to compare samples from affected and normal Keeshonds with mutated genes known to cause hyperparathyroid disease in humans. The three genes known as “candidate” genes were checked but none matched the pattern of abnormality found in the genome of affected Keeshonds.

In 2005 a parallel project commenced in the USA as a result of a Keeshond being treated at Cornell and Dr. Richard Goldstein agreed to investigate the disease. His involvement was as a result of treating a 6 year old Keeshond Kylie. His owner Cathy Bosnic has worked tirelessly to encourage owners in the States to participate in the study and has been a driving force in working closely with Dr. Goldstein. They started to collect samples from known affected animals and close relatives. Again the “candidate genes” (those identified in humans) were eliminated and at the time of the genome scan he had over 200 samples with over 50 from affected animals.

By 2005 it became apparent that our research in the UK would need a second funding award to continue. Again a bid was submitted to the KCCT supported by both the North of England Keeshond Club and The Keeshond Club. In January 2006 £25,000 was awarded and along with the £1,500 pledged by each of the clubs gave a total of £28,000 for the vital work. This second bid included looking for the genetic cause of primary epilepsy as well because we had been steadily collecting samples from dogs diagnosed with primary epilepsy alongside the PHPT collection. I will return to the epilepsy research later.

Research is never without its frustrations and we had to wait for 6 months to receive the £25,000! Once Richard announced that the gene and mutation had been located, our research into PHPT had to cease. The good news however, is that Barbara is now working closely with Richard at Cornell and our samples will help to further confirm the accuracy of the test.

How was the Gene found?

In collaboration with Dr. Kerstin Lindblad-Toh and Dr. Claire Wade of the Broad Institute of Harvard and MIT the samples collected by Dr. Goldstein were analysed.  It was possible to search the entire canine genome and to identify the locus or region that most likely contained the gene mutation causing the disease. This new technology was able to assess tens of thousands of markers throughout the DNA to find an association between these markers and the disease. The result of this was that the entire DNA was narrowed down to just three genes. Richard continued to closely examine these genes until it became apparent that only one included markers that were highly associated with the disease. Within that gene was an abnormality that is now being tested for.

Autosomal dominant mode of inheritance

The research has confirmed that the mode of inheritance of PHPT in the Keeshond is a dominant trait. A number of known inherited diseases in the dog are autosomal recessive with the designation of an animal being “clear”, “affected” or “carrier”. This is what primary epilepsy is thought to be.

With an autosomal dominant mode of inheritance, an abnormal gene from one parent is all that is needed to pass the disease onto the progeny. It is not necessary for the other parent to have the gene. If a Keeshond with the defective autosomal dominant gene for PHPT is used for breeding, then 50% of their offspring will also have the gene while the other 50% will not. If two dogs with the defective gene are mated then 50% will have one copy of the defective gene, 25% will have two copies of the normal gene and 25% will have two copies of the defective PHPT gene. Research has suggested that embryos with two copies of the defective gene may not survive in utero (within the uterus), resulting in smaller litter sizes for this type of breeding.

There is no “carrier” state with an autosomal dominant mode of inheritance. Either an animal has the gene or it does not. The test devised at Cornell gives the designation of either “positive” for the PHPT gene or “negative” for the gene.

The UK Kennel Club web site has an excellent article, ‘Dog to DNA’, which explains the basic genetics of the dog and the differences between autosomal dominant and recessive genes.

Will all dogs with the gene develop PHPT?

The disease is known to affect older dogs with the mean age in earlier research being 10 years. More recently animals as young as 6 years have been diagnosed. The disease affects older Keeshonds with what is known as “age dependant penetrance”. The DNA test does not identify the clinical disease but rather indicates the presence of the gene. This indicates the genetic potential to develop the disease later in life. This is a cancer causing gene and most animals that carry it will go onto develop the disease if they live long enough.

How accurate is the test?

Some people in the States have questioned the validity of a test and as a result Richard has fully explained the situation via the various web based breed discussion lists.

The uncertainty seemed to arise out of the finding that 7% of older dogs sampled who had no clinical signs proved positive for the gene. For this reason they questioned whether the test threw up false positives.

Richard explained that it is a linkage test with 100% association to the disease PHPT. This means that the test is for a site in the DNA either within the gene itself or very, very close to the gene, so it is linked to the disease.

The sample size of over 200 dogs with over 50 affected was much larger than generally used to validate a genetic test. He was able to confirm that all samples from dogs diagnosed with the disease tested positive for the gene. Statistically this is consistent with a zero distance between the mutation causing the disease or 100% accuracy. He also confirmed that based on the samples sent into the study the vast majority of dogs that live long enough will get the disease (over 95% if they live until they are twelve). These numbers could change as more dogs are tested.

When our UK research was halted, our researcher Barbara made contact with Richard Goldstein and as a result he offered to test all our UK samples. We have approximately the same number of samples and affected samples so this will effectively double the number of animals included in the survey.

Barbara converted all our blood samples into stabilized DNA and these were shipped to the USA. Our samples were run in a blind fashion so their status for the disease was unknown at the time of testing. The results are now back at Cambridge and again dogs previously diagnosed by the parathormone test have been confirmed positive for the gene , along with others who were not seen to present clinical symptoms, Our samples have doubled the sample size and further confirmed the accuracy of the test.

How to get dogs tested?

The test is now commercially available via Richard and his website provides detailed information on both the condition and also the testing procedure.

The site contains a series of articles written by Cathy Bosnic, a full explanation of the research, a “questions and answers” section and all the forms and protocols needed to submit a blood samples for analyses Bloods can be sent from outside the USA and the protocol for importing the blood into the USA is also explained.

Testing is an investment in the future. Many American and Canadian breeders have been posting the results of their dogs test on the various breed discussion lists. If both the sire and dam of a litter prove “negative” for the PHPT gene then by definition their progeny will also be gene negative as they cannot inherit what isn’t there. This means that the entire litter do not need to be tested. In time the need to test will reduce as the status of ancestors is known and recorded.

In the USA some breeders are sending in samples as a group and in doing so are gaining discounts as well as saving on the cost of shipping. I believe this is also happening in Germany. Barbara is happy to coordinate dispatch of samples to Cornell to enable British breeders and owners to get the group discount. I am happy to advise on the documentation needed and procedure.

Samples tested from other countries must follow the guidelines revised on July 6, 2006 by the USDA. The veterinarian needs to include a declaration statement to allow passage of the sample without a permit.

The Epilepsy research continues

So what about epilepsy? As some of you will know in 2004 I circulated paperwork on the English Epilepsy project with the purpose of collecting DNA samples from dogs diagnosed with primary epilepsy. Barbara checked all the paperwork for accuracy and the questionnaire was circulated along with protocols for sending blood to Cambridge. I am grateful to the people in various countries who kindly circulated the information to Keeshond owners and breeders.

As a result we have received samples from Australia, the USA and Europe to add to the English ones and it is as a result of those peoples commitment that the epilepsy portion of the English bid was successful.

As a result of the joint collaboration with Dr. Richard Goldstein and Dr. Barbara Skelly we are now in a position to analyse these samples in the States using the same package at the Broad Institute that identified the gene responsible for PHPT.

It has always been accepted that inherited epilepsy in the Keeshond is as a result of an autosomal recessive mode of inheritance but more recent work suggests that we are not looking at a simple gene but more likely more than one gene with a single gene of major effect.

This is an exciting development and we can all hope that the analysis will be as successful as the PHPT has been. Dr. Richard Goldstein and his team have shown that it is possible to map disease traits rapidly in the Keeshond so it makes sense to take advantage of the new techniques at our disposal and make sure that epilepsy also becomes a disease of the past.

If anyone with a dog diagnosed with primary epilepsy would like to participle we would be happy to accept a sample. I can supply the information privately. If you would like to participate in the Epilepsy Survey you can contact Barbara as follows:

Barbara J Skelly MA VetMB PhD CertSAM DACVIM DECVIM MRCVS
Department of Veterinary Medicine
University of Cambridge
Madingley Road
Cambridge CB3 0ES
Tel: (01223) 337621/337669
Fax: (01223) 330848
e-mail: Dr. Barbara Skelly

Our responsibility

As a breed we are so lucky to have such committed and talented researchers on our side. Being a numerically small breed we need to take care of our future. At the end of the day I wouldn’t be writing this article if it were not for the Keeshond dog and our lives would be the poorer without them.

 

Anja aged 8 weeks – they are our future

We have seen drastically falling registrations across many countries. In the UK annual registrations have been under 100 since the year 2000.

Standing up for health does not win friends in some quarters but at the end of the day the welfare of the breed is paramount and every dog bred should have the right to be free from inherited disease and unnecessary suffering.

 We can only eradicate inherited disease if everyone is open an honest. We could make PHPT history in a very short time but only if the welfare of the dogs is put before self interest. Our motto should be “dogs come first”! 

DNA is an exact science and ends the years of guesswork and finger pointing. Don’t let this wonderful opportunity be wasted.

 I have lived with a Keeshond for nearly 50 years and couldn’t imagine life without them. They give their all – we owe it to them to give ours.

 

 Acknowledgements

Dr. Richard Goldstein DVM DACVIM DECVIM is an associate professor of small animal medicine at Cornell University’s College of Veterinary Medicine.

I would like to thank Dr. Richard Goldstein and Dr. Barbara Skelly for all their hard work and dedication on behalf of our breed. In compiling this article I have referred to recent posts from Richard as well as his web site to explain the identification of the gene and the test availability.

I am grateful to Barbara for her unstinting support and her patience over the past 4 years and for helping me gain some understanding of genetics! I would also like to thank her for proof reading this article for me.

References

Berger B, Feldman EC. (1987) Primary hyperparathyroidism in dogs: 21 cases (1976-1986). Journal of the American Veterinary Association;191(3): 350-6.

Weir EC, Norrdin RW, Barthold SW, Meuten DJ, Pond MJ, Insogna KL. (1986) Primary hyperparathyroidism in a dog: biochemical, bone histomorphometric, and pathologic findings. J Am Vet Med Assoc. 189(11): 1471-4.

© Jane Saunders – May 2007

 

Keeshond Primary Hyperparathyroid (PHPT): Genetic Test

The availability of a genetic test to determine whether an animal has the gene for PHPT has given us an unprecedented chance to rid our beautiful breed of this disease. With the knowledge we now have Primary Hyperparathyroid disease can soon become history.

Why test for the PHPT gene?

The disease is known to affect older dogs with the mean age in earlier research being 10 years. More recently animals as young as 6 years have been diagnosed. The disease affects older Keeshonds with what is known as “age dependant penetrance”. The DNA test does not identify the clinical disease but rather indicates the presence of the gene. This indicates the genetic potential to develop the disease later in life.

Will all dogs with the gene develop PHPT?

This is a cancer causing gene and most animals that carry it will go onto develop the disease if they live long enough.

If your dog has been tested you will be able to know whether it is “positive” or “negative” for the PHPT gene.

If both parents have been tested and are negative then their offspring will also be negative by descent because they cannot inherit what their parents do not have. This is also known as “hereditarily negative”.

How much will it cost?
AHDC laboratory at Cornell has issued a new submissions form with effect from January 2nd 2012

The test cost is $90.00 per sample, plus an accession fee of $2 making a total of $92 ber dog.

If a group of owners can arrange for bloods to be  sent together,  the shipping cost can be shared. Co-operation with payments can also save on the cost of an international money draft if one is requested to meet the total cost of all samples.

Shipping

The preferred method is by courier. The cost of sending a sample via FedEx priority service is currently £51.85 for a 0.5 kg package. Other couriers are available.

Hints and tips on getting your dog tested and completing the paperwork

The instruction sheet for submitting blood to the Cornell Animal Health Diagnostic centre can be found on Dr Goldstein’s laboratory site. Get Instruction Sheet

Before visiting your veterinary Surgeon, you will need to download and complete the following  submission form from the Cornell Diagnostic Centre web site. You can also download it below.

  1. The Cornell Animal Health Diagnostic Centre (AHDC): General Submission Form, which is to be completed by the vet who draws the blood samples. (updated 2/1/2012)
    Get Form

Adobe Acrobat Reader will be required to view/print the protocol and  submission form. If needed, this free software can be downloaded from here.When completing these forms, please use black ink and when inserting the date of birth in the ‘Dog Information’ section of the second form the American date format(MM/DD/YY) should be used. Take this form with you when you visit your vet to have your dogs blood sample taken, together with A five generation pedigree should also be included, which can be printed from here.

When completing your paperwork ensure that your vet puts both a practice fax number and/or e-mail address to enable the Cornell ADHC to return your results as quickly as possible.

Please ask the submitting vet to list their e-mail address  if they wish to receive the test results by e-mail, also ensure you list your own e-mail address, if you have one.

Failure of your vet to provide either a practice fax number and/or e-mail address will result in your test result(s) being posted via US mail, with the inevitable delay of at least several days.

Declaration of contents

Please ask you vet to write a short statement stating that it is “non infectious canine blood” and enclose one on the inside of the package and also one on the outside of the package. Also, with FedEx, write NCV, which means “No Commercial Value” and also “Serums – Non-Hazardous” Basically they want to know that the blood is not infected with anything contagious. Any Queries should be sent to Lisa at Cornell AHDC.

All completed paperwork, together with your payment should be left with your vet who will enclose and send them with the sample. It is advisable to ask your vet to keep a copy of the paperwork for your record.

Payment of the genetic test

Payment of the genetic test is required in US$ so suitable payment will need to be arranged.

There are several ways to send payment in US$

International money draft

      The cost of arranging an international money draft varies between banks but there is an administration charge of £15 – £25 for producing the cheque. You will need to consult your own bank or building society to find out what they charge. Allow a few days for them to process it.

Credit/debit card

        The AHDC will accept payment via ad credit/debit card. The charge will be based on the exchange rate on the day of transfer plus an additional charge of 2.5% -3.00% of the total transaction.The form for paying by credit/debit card can be downloaded

here

      .
      Please ensure that all the relevant details are completed and the this is attached to your AHDC submission form.The transaction will be shown on your next available statement along with the handling charge.With the rise in credit card fraud companies may refuse to authorise a payment to another country if it is outside your normal pattern of spending. To avoid any possible difficulties it is advisable to inform your bank/credit card company that you will be making a payment to the AHDC Cornell University USA.

At present there is no online payment system

Please don’t forget that payment must now accompany the submitted sample(s).

If you have any questions regarding these methods of payment, please contact Dr. Belinda Thompson, by e-mail or phone 001 607-253-3908

Sending your sample to Cornell?

Details for posting can be found on the laboratory instruction sheet.

If you have any queries at all please do not hesitate to contact me. Either by e-mail or phone on 023 8073 5494

© Jane Saunders – updated July 2012

 

DNA Screening – PHPT- Primary Hyperparathyroidism in the Keeshond

The test for PHPT is now commercially available via Richard Goldstein DVM and his website provides detailed information on both the condition and also the testing procedure.

PHPT in the Keeshond has an autosomal dominant mode of inheritance. The test confirms whether a dog is ‘positive’ for the PHPT gene or ‘negative’ for the PHPT gene. There is no carrier state in a dominant mode of inheritance.

If dogs that are positive for the PHPT gene are bred from they will pass a defective gene on to approximately half of their offspring if one parent only is positive, or ¾ of the offspring if both are positive. Research has shown that two positive parents can produce reduced litter sizes because there are some that do not survive to birth. Only one positive parent is needed to pass the gene onto their progeny. The disease is known to have ‘age dependant penetrance’ which means that most or all animals that carry the gene will go onto develop the disease if they live long enough.

The DNA test does not identify the clinical disease but rather indicates the presence of the gene. This indicates the genetic potential to develop the disease later in life.

If both sire and dam are negative for the defective gene then the progeny will be hereditarily negative/negative by descent as they cannot inherit what their parents did not have.

The UK Kennel Club website has an excellent article, ‘Dog to DNA’ explaining basic genetics of the dog and the difference between autosomal dominant and recessive genes.

The Keeshond Club PHPT Open Registers, listing those Keeshonds who have tested gene negative or positive as well as those who are hereditarily negative (negative by descent) are available here.

© Jane Saunders – December 2007