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

33

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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