Jonathan Beever, PhD University of Illinois November 2, 2006

1. 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 • 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 • 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 • 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. PROBAND 3 3 5 1 2 1 4 1 4 4 3 1 1 1 homozygosity analysis

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 • 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) • 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

19. 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

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 • 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