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Breed Improvement Through DNA Testing By Nathan Dewsbury, MSc Breed stewards and enthusiasts all care about the health and greater well being of their animals. All animal lovers want to provide the best for their pets in the way of the best food, the best health care, and the best toys available. Breed stewards seek to go beyond the external applications and instead look internally at the genetic aspect of pampering their animals in the form of genetic improvement. It’s this fundamental desire of providing the best for another life form other than your own that leads breed stewards to take on the task of breeding. A steward, who is already knowledgeable in their breed of choice, is always aware of the genetic health conditions of their breed and serves as the most commonly sought aspect of improvement. This increased search for greater canine health is driving the development for better tools, in the form of DNA testing that is going to give breed stewards a “leg up” in breed development. So What is Merle and Why is it a Problem? In the domestic dog merle coloration is found in a number of breeds (Australian Shepherd, Cardigan Welsh Corgi, Shetland Sheepdog, Collie, Great Dane, Catahoula Leopard Dog) recognized by major canine registries. It is in these breeds, where the merle phenotype (coloration) is common place, that stewards are faced with a phenotype that is linked to major physical defects most notable in eye sight and hearing impairment. Both of which defects drastically affect the daily life and the over all well being of the affect animal(s) from birth. Merle coloration is presented as patches of diluted pigment interspersed with normal melanin coloration of that breed, which usually shows up as spots (Figure 1). This merle phenotype is the result of a mutation that has resulted in additional genetic information being inserted into the normal (wild type) gene called SILV, which is found on chromosome 10. This additional genetic code is the result of a class of Retrotransposon, referred to as a jumping gene, that can cut and past it’s self any where in the canine genome, thus altering the function and expression if inserted in a gene. The SILV gene itself has a central role in pigmentation with significant expression of the gene in the skin and the eye. This functional role is especially crucial as lack of pigmentation in the hair inside of the ear canal results in deafness. Most individuals know the association of pigmentation and its link to genetic abnormalities on a general level. Skin color in humans is a good example. We know that the lighter your skin complexion the greater your possibilities to develop certain skin cancers if unprotected under prolonged exposures in the sun. Merle pigmentation is determined during embryo development, not a result of an onset condition that could possibly be treated or reversed. The merle mutation is responsible for developmental abnormalities that affects cardiac, ophthalmic, auditory, and reproductive systems in these domestic canines. In the case of the merle mutation, the developmental issues exhibited by affected canines share similar abnormalities observed in the human auditory pigmentation disorder Waardenburg syndrome (WS). Both merle and WS can

Canine Merle Mutation Article

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Breed  Improvement  Through  DNA  Testing  By  Nathan  Dewsbury,  MSc  

    Breed  stewards  and  enthusiasts  all  care  about  the  health  and  greater  well  being  of  their  animals.  All  animal  lovers  want  to  provide  the  best  for  their  pets  in  the  way  of  the  best  food,  the  best  health  care,  and  the  best  toys  available.  Breed  stewards  seek  to  go  beyond  the  external  applications  and  instead  look  internally  at  the  genetic  aspect  of  pampering  their  animals  in  the  form  of  genetic  improvement.  It’s  this  fundamental  desire  of  providing  the  best  for  another  life  form  other  than  your  own  that  leads  breed  stewards  to  take  on  the  task  of  breeding.    A  steward,  who  is  already  knowledgeable  in  their  breed  of  choice,  is  always  aware  of  the  genetic  health  conditions  of  their  breed  and  serves  as  the  most  commonly  sought  aspect  of  improvement.  This  increased  search  for  greater  canine  health  is  driving  the  development  for  better  tools,  in  the  form  of  DNA  testing  that  is  going  to  give  breed  stewards  a  “leg  -­‐up”  in  breed  development.    So  What  is  Merle  and  Why  is  it  a  Problem?       In  the  domestic  dog  merle  coloration  is  found  in  a  number  of  breeds  (Australian  Shepherd,  Cardigan  Welsh  Corgi,  Shetland  Sheepdog,  Collie,  Great  Dane,  Catahoula  Leopard  Dog)  recognized  by  major  canine  registries.  It  is  in  these  breeds,  where  the  merle  phenotype  (coloration)  is  common  place,  that  stewards  are  faced  with  a  phenotype  that  is  linked  to  major  physical  defects  most  notable  in  eye  sight  and  hearing  impairment.  Both  of  which  defects  drastically  affect  the  daily  life  and  the  over  all  well  being  of  the  affect  animal(s)  from  birth.  

    Merle  coloration  is  presented  as  patches  of  diluted  pigment  interspersed  with  normal  melanin  coloration  of  that  breed,  which  usually  shows  up  as  spots  (Figure  1).  This  merle  phenotype  is  the  result  of  a  mutation  that  has  resulted  in  additional  genetic  information  being  inserted  into  the  normal  (wild  type)  gene  called  SILV,  which  is  found  on  chromosome  10.  This  additional  genetic  code  is  the  result  of  a  class  of  Retrotransposon,  referred  to  as  a  jumping  gene,  that  can  cut  and  past  it’s  self  any  where  in  the  canine  genome,  thus  altering  the  function  and  expression  if  inserted  in  a  gene.  The  SILV  gene  itself  has  a  central  role  in  pigmentation  with  significant  expression  of  the  gene  in  the  skin  and  the  eye.  This  functional  role  is  especially  crucial  as  lack  of  pigmentation  in  the  hair  inside  of  the  ear  canal  results  in  deafness.  Most  individuals  know  the  association  of  pigmentation  and  its  link  to  genetic  abnormalities  on  a  general  level.  Skin  color  in  humans  is  a  good  example.  We  know  that  the  lighter  your  skin  complexion  the  greater  your  possibilities  to  develop  certain  skin  cancers  if  unprotected  under  prolonged  exposures  in  the  sun.  Merle  pigmentation  is  determined  during  embryo  development,  not  a  result  of  an  onset  condition  that  could  possibly  be  treated  or  reversed.  The  merle  mutation  is  responsible  for  developmental  abnormalities  that  affects  cardiac,  ophthalmic,  auditory,  and  reproductive  systems  in  these  domestic  canines.  In  the  case  of  the  merle  mutation,  the  developmental  issues  exhibited  by  affected  canines  share  similar  abnormalities  observed  in  the  human  auditory-­‐pigmentation  disorder  Waardenburg  syndrome  (WS).  Both  merle  and  WS  can    

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   display  a  range  of  physical  changes  as  a  result  of  the  genetic  changes.  Alterations  include  the  appearance  of  blue  eye(s),  eye  shape  deformities,  blindness,  levels  of  deafness,  and  cardiac  valve  problems.  

 

 Figure  1.  Demonstrates  the  three  possible  phenotypes  associated  with  the  semi-­‐dominant  inheritance  of  the  merle  phenotype.  The  dog  on  the  left  contains  a  double  merle,  in  the  middle  is  a  normal  (wild  type)  dog,  and  the  dog  on  the  right  inherited  a  single  merle  gene.  Image  taken  from  Genomics  of  Disease  book  

chapter  (Stadler  Genomic  Symposia  Series)  by  J.P.  Gustafson,  March.  2008    

How  is  the  Merle  Mutation  Passed  On?      

Knowing  the  deleterious  effects  associated  with  the  merle  genotype  one  might  ask  how  is  it  that  the  merle  pattern,  hence  the  merle  mutation,  is  still  present  in  certain  breeds?  Breeding  practices  alone,  no  matter  how  stringent  the  selection  criteria,  could  not  selectively  breed  out  the  mutation  with  out  genetic  testing.  This  is  the  primary  reason  for  the  continual  occurrence  of  the  merle  mutation  after  the  divergence  of  canine  breeds  and  still  today.  In  addition  to  the  possible  expressed  phenotypes  (Figure  1)  the  situation  arises  when  a  dog  can  be  genotypic  merle  yet  does  not  present  a  traditional  merle  pattern  phenotype.  These  dogs  are  coined  a  cryptic  merle.  These  dogs  appear  to  have  solid  coat  colors  or  multiple  solid  colors  instead  of  the  obvious  merle-­‐spotted  appearance.  It  was  assumed  that  these  dogs  were  solid  or  non-­‐merle  dogs  and  thus  would  be  incorporated  into  the  breeding  population  only  to  perpetuate  the  merle  mutation.  The  only  way  to  fully  know  the  genotype  of  a  dog  is  through  DNA  testing.      

The  merle  mutational  trait  is  inherited  in  an  autosomal  semi-­‐dominant  fashion  as  demonstrated  by  the  hypothetical  breeding  illustrated  in  Figure  2  below.  Using  a  Punett  square  model,  we  can  see  in  Figure  2,  that  the  presence  of  a  merle  genotype  parent  alone  is  not  enough  to  produce  all  merle  offspring.  The  semi-­‐dominance  is  expressed  in  terms  of  the  presence  of  a  single  merle  allele  that  when  inherited  will  be  expressed  dominantly  in  the  phenotype  of  that  offspring,  but  in  the  absence  of  the  mutant  allele  the  wild  type  phenotype  will  be  expressed  dominantly  (Figure  2,  Breeding  B  &  D)  even  if  one  or  both  parents  are  carriers.    Inheritance  of  a  single  merle  locus  (heterozygous)  usually  is  not  associated  with  any  health  effects        

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for  that  animal.  In  all  merle  breeds  that  are  genotypic  double  merles  (homozygous)  for  the  merle  locus  will  result  in  subleathal  effects.  This  genotype  is  responsible  for    the  multiple  abnormalities  associated  with  the  incorporated  merle  mutation.  An  exception  to  the  resultant  double  merle  effects  is  found  in  the  Catahoula  Leopard  Dog.  The  merle  mutation  has  shown  to  behave  differently  with  this  breed,  which  shows  less  susceptibility  to  exhibit  the  negative  effects  of  merling  when  compared  to  other  breeds.  

 

     

Figure  2.  Demonstrates  the  breeding  possibilities  to  illustrate  the  semi-­‐dominant  inheritance  pattern  of  the  merle  gene  found  in  the  domestic  canine.    

 How  Can  DNA  Testing  Help?    

Once  only  imaged  by  breeders  as  an  act  of  science  fiction,  DNA  diagnostic  tests  are  becoming  increasingly  available  through  DNA  testing  services  and  may  soon  become  the  norm  in  future  breeding  programs  or  even  in  pet  selection.    

The  use  of  DNA  diagnostic  tests  have  been  implemented  for  a  number  years  and  are  primarily  conducted  or  initiated  by  veterinarians  for  the  diagnosis  of  ailments  and  inflictions  such  as  canine  epilepsy  or  Parvovirus.  Older  diagnostic  methods  entailed  multiple  complex  steps  that  lengthened  reporting  of  results  yielding  semi-­‐specific  results.  Most  diagnostic  tests  are  implemented  in  the  detection  of  an  on-­‐set  health  aliment.  New  innovations  in  research  have  enabled  the  development  of  more  streamline  testing  protocols  yielding  specific  results  with  fewer  steps.  An  additional  aspect  brought  about  by  innovation  is  the  use  of  DNA.  The  ability  of  a  diagnostic  approach  to  screen  DNA  is  now  preventing  abnormal      health  development  via  detected  genetic  conditions  in  parental  animals  that  would  have  caused  or  led  to  health  defects.    The  advantages  of  DNA  diagnostics  are  

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demonstrated  by  the  availability  of  diagnostic  tests  to  determine  parentage,  presence  of  specific  disease  markers,  prediction  of  coat  coloration  and  length  are  to  name  a  few  offered  by  companies  outside  of  the  veterinary  care  system.    

 Now  stewards  faced  with  the  merle  phenotype  have  yet  another  tool  to  

further  breed  health  progression  with  the  development  of  the  Merle  Test,  a  DNA  diagnostic  test,  for  the  detection  of  the  merle  mutation.  Prior  to  the  development  of  merle  testing,  such  enacted  breeding  practices  to  diminish  the  merle  occurrence  and  detrimental  affects  entailed:  no  merle-­‐to-­‐merle  breeding,  breeding  merle  to  only  solid  colored  dogs,  avoidance  of  predominant  white  dogs  in  breeding  programs,  and  scatter  breeding  to  solid  colored  dogs  every  two  or  three  generations  for  certain  breeds.  These  practices  have  served  breeders  well,  but  they  take  a  large  investment  in  time,  breeding,  and  experience  to  prove  the  level  of  merle  expression.  This  leaves  error  of  selection  and  opportunity  for  vast  improvement  in  way  of  breed  improvement.  

 Dr.  Leigh  Anne  Clark,  Texas  A&M  canine  geneticist  developed  the  licensed  

Merle  Test  during  her  research  in  determining  merle  patterning  in  the  domestic  dog.  This  straightforward  DNA  diagnostic  test,  provided  by  GenMark’s  Merle  Test,  determines  the  presence  of  the  merle  gene  and  it’s  level  of  genetic  dominance.  A  dog  owner  wishing  to  know  the  genotype  of  their  merle  dog  or  those  associated  with  merle  dogs  have  to  simply  take  a  mouth  swab  of  the  animal(s)  of  interest  and  send  the  swab  back  to  GenMark  for  testing  and  analysis.  Using  a  polymerase  chain  reaction  (PCR)  method  allows  GenMark  to  amplify  the  region  of  chromosome  10  that  encodes  for  the  SILV  gene,  which  will  either  contain  a  mutational  insert  or  not.      The  results  gained  from  this  testing  informs  the  owner(s)  to  the  level  of  merle  expression  in  their  animals  as  carriers,  non-­‐carriers  or  genetically  dominant  (double  merle)  for  the  merle  locus.      

 Armed  with  conclusive  evidence  allows  for  more  informative  breeding  

practices  and  improved  mate  selections  in  an  effort  to  remove  the  merle  mutation  from  breeding  populations  of  domestic  canines.  The  decreasing  presence  of  the  merle  mutation  will  lead  to  an  increase  in  breed  health.  Eventual  removal  through  DNA  testing  and  selective  breeding  practices  will  result  in  breed  improvement  on  the  basis  of  removing  an  un-­‐advantageous  genetic  source  that,  when  present,  only  introduces  defects  in  important  functional  systems  of  life.    Advances  in  DNA  diagnostics,  through  further  understanding  of  the  canine  genome,  in  the  form  of  genetic  inheritance  testing  for  animals  is  proving  to  be  a  real  world  tool  available  to  people.  DNA  diagnostics  has  the  ability  to  translate  scientific  research  into  functional  applications  to  aid  breed  stewards  in  their  efforts  toward  canine  health  improvement.