Tibial Hemimelia TH and Pulmonary Hypoplasia with Anasarca PHA _____________________ What are they,

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Tibial Hemimelia (TH) and Pulmonary Hypoplasia
with Anasarca (PHA)_____________________What
are they, where are they and how are they relevant

• Jonathan Beever, PhD
• University of Illinois
• November 2, 2006

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tibial hemimelia (th)

• skeletal defects
• failure of pelvic fusion abdominal hernia
• shortened or absent tibia severe distortion of
  rear leg structure
• failure of proper neural tube closure exposure
  of brain or spinal tissue
• other defects
• cryptorchidism, failed Mullerian duct development
• invariably lethal
• calves may be live born fail to thrive,
  euthanized

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background

• recognized in Galloway cattle in early 70s (Ojo
  et al. 1974)
• documented sire test/selection program in UK
• genetic inheritance
• Reported in in Shorthorn cattle in 2000 (Lapointe
  et al. 2000)
• 3 of 6 calves reported of Canadian origin
• ancestry common among all calves

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genetics

• unaffected parents (i.e., normal is dominant)
• equal frequency among sexes
• pedigree analysis reveals common ancestry on both
  sides of pedigree
• expected ratios of offspring among matings
  between carrier (heterozygous) parents
• 31 ratio of normal to affected offspring
• recessive Mendelian inheritance
• animals homozygous for defect (mutation) are
  affected
• both parents of affected calves must be carriers

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potential impact

• worldwide
• putative common ancestor is early Irish import
• one of few direct imports extensive use
• circa 1975 multiple generations of dispersion
• multiplied in US exportation of germplasm
• US (2004 perspective)
• more than half of the top 10 sires for number of
  Shorthorn registrations are putative carriers
• popular club calf sire is suspected carrier
• estimated 80,000 units of semen sold
• In 2005, 21 of 24 black composite AI sires
  offered by a single vendor are tested as carriers

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how to find the defective gene

• identification of appropriate pedigree/population
  material
• collect DNA samples
• 60 individuals of known genotype status
• within nuclear families
• genetic marker screening
• even distribution/coverage across genome
• panel of 263 markers
• prioritize chromosomes for analysis
• comparative biology/genomics

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homozygosity analysis
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comparative genomics
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mutation screening

• complete DNA sequencing of causative gene
• 140,000 base pairs
• resequencing of animals of known genotype
• normal, carrier and affected
• no variation in DNA sequence that was consistent
  between all known animals
• inability to resequence portion of gene in
  affected calves
• significant portion (30) of gene absent in
  affected calves

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Figure 1. Photograph demonstrating the DNA-based
test for tibial hemimelia (TH). The DNA from
each of ten individuals was used to determine
their TH status by PCR amplification of the
normal chromosome segment and the mutated
chromosomal segment simultaneously. Animals in
lanes 1, 6 and 9 are homozygous normal due to the
presence of only the DNA segment representing the
normal chromosome. Animals in lanes 2, 4 and 8
are homozygous for the chromosome with the
deletion mutation causing TH, indicating that the
samples were taken from affected calves. Animals
in lanes 3, 5, 7 and 10 possess both DNA segments
indicating that they are heterozygous or carriers
of the mutation.
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validation

• blind testing of 45 animals of known status
• 100 accurate
• random testing of 300 phenotypically normal
  individuals
• none homozygous for mutation
• testing of 7 known sires confirmed by ASA genetic
  defect policy
• only 6 of 7 genotype as carriers

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resolution

• different/inconsistent phenotype?
• Pulmonary Hypoplasia with Anasarca (PHA)
• all affected calves from inconsistent sire
  genotype as homozygotes for identified mutation
• all affected calves parentally verify to sire
• except for DNA markers adjacent to causative gene
• 2nd mutation complete deletion of gene
• complete deletion of 4 genes (460,000 bp)
• very rare frequency as compared to first

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curiosities

• selection paradox
• carriers are the best
• is there a quantitative measure to define best?
• non-pathological manifestation in heterozygotes?
• structural differences in hindquarters
• remember gene function
• perstistance and selective increase in the
  breeding population over time
• almost impossible to dilute

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pulmonary hypoplasia with anasarca (PHA)

• pulmonary hypoplasia
• absent or near absence of lungs
• normal cardiovascular system
• anasarca
• tremendous fluid accumulation in affected calves
• lack of lymphatic development
• absence of lymph duct and nodes, athymia
• invariably lethal
• all near term calves born dead
• other
• early embryonic lethal increased open rate
  after confirmed pregnancy

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genetics

• unaffected parents (i.e., normal is dominant)
• equal frequency among sexes
• pedigree analysis reveals common ancestry on both
  sides of pedigree
• deficiency of affected calves given suspected
  frequency
• recessive Mendelian inheritance
• affected pedigrees in both Shorthorn and Maine
  Anjou breeds

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potential impact

• putative common ancestor is early French or
  Canadian import
• circa 1975 multiple generations of dispersion
• multiplied in US
• 40 of 121 popular club calf sires are carriers
• potential for phenotypic selection in the
  carriers
• 80 of sons in AI service that are sired by a
  popular carrier club calf sire are carriers

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mutation screening

• complete DNA sequencing of causative gene
• resequencing of animals of known genotype
• normal, carrier and affected
• single missense mutation common to modern
  Shorthorn, Maine Anjou and composite cattle

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validation

• blind testing of 144 animals of known status
• 100 accurate
• random testing of 1000 phenotypically normal
  individuals
• none homozygous for mutation
• 4 suspect sires test normal
• insufficient evidence of their status

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risk assessment

• do you care?
• methods to assess risk
• pedigree analysis
• do your pedigrees contain suspect individuals?
• including modern sires that have been tested
• diagnostic screening
• random testing within your herd
• suspect pedigree representation

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pedigree assessment

• at what point in a pedigree doesnt it matter
  anymore?
• how many generations?
• (1/2)n probability of carrier
• n number of generations between known carrier
  and individual in question
• 1 generation 50
• 3 generations 12.5
• 8 generations 0.4
• additive consider all suspect individuals with
  independent paths to individual

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breeding management

• education is key
• understand the possibilities desired outcome
• do nothing vs. kill em all
• up to individual breeders vs. mandatory testing
  and culling of all carrier animals
• accurate identification of carriers
• selective vs. comprehensive testing programs
• voluntary vs. mandatory

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what to test

• expense vs. outcome
• low cost no affected calves born
• sires only no affected calves born to TH-Free
  sires
• moderate cost on the road to elimination
• sires, herd matriarchs and annual replacement
  heifers
• highest cost complete management
• all animals in the herd
• does not imply elimination, only management

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acknowledgements

• Charles P. Hannon, DVM
• Nick Steinke
• Brandy Marron
• Geri Thurneau
• USDA CSREES/ARS LGSI