Tibial Hemimelia (TH) and Pulmonary Hypoplasia with
Anasarca (PHA)_____________________
What are they, where are they and how are they relevant
Jonathan Beever, PhDUniversity of IllinoisNovember 2, 2006
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
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
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 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
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
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
PROBAND
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homozygosity analysis
comparative genomics
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
THnormal
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.
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
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
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”
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
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
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
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
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
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
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
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
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
acknowledgements
Charles P. Hannon, DVM Nick Steinke
Brandy Marron Geri Thurneau
USDA CSREES/ARS – LGSI