1. Jonathan Beever, PhD University of Illinois November 2, 2006
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

2. tibial hemimelia (th) skeletal defects other defects invariably lethal
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

3. background
recognized in Galloway cattle in early 70’s (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

6. genetics recessive Mendelian inheritance
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
3:1 ratio of normal to affected offspring
recessive Mendelian inheritance
animals homozygous for defect (mutation) are affected
both parents of affected calves must be carriers

7. potential impact worldwide US (2004 perspective)
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

8. 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

10. homozygosity analysis
PROBAND 3 5 1 2 4

11. comparative genomics

12. 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

13. TH
normal 1 2 3 4 5 6 7 8 9 10
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.

14. 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

15. resolution 2nd mutation – complete deletion of gene
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

16. 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”

17. pulmonary hypoplasia with anasarca (PHA)
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

19. genetics recessive Mendelian inheritance
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

20. 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

21. 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

22. 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

23. 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

24. pedigree assessment
at what point in a pedigree doesn’t 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

25. breeding management education is key do nothing vs. “kill ‘em all”
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

26. 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

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