Tibial Hemimelia TH and Pulmonary Hypoplasia with Anasarca

Tibial Hemimelia (TH) and Pulmonary Hypoplasia with Anasarca (PHA) ___________ What are they, where are they and how are they relevant Jonathan Beever, Ph. D University of Illinois November 2, 2006

tibial hemimelia (th) n skeletal defects n n other defects n n 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 cryptorchidism, failed Mullerian duct development invariably lethal n calves may be live born – fail to thrive, euthanized

background n recognized in Galloway cattle in early 70’s (Ojo et al. 1974) ¨ documented n n sire test/selection program in UK genetic inheritance Reported in in Shorthorn cattle in 2000 (Lapointe et al. 2000) ¨ 3 n 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 n n 3: 1 ratio of normal to affected offspring recessive Mendelian inheritance n n animals homozygous for defect (mutation) are affected both parents of affected calves must be carriers

potential impact n worldwide ¨ putative common ancestor is early Irish import n n 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 ¨ n ¨ 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 n identification of appropriate pedigree/population material n n collect DNA samples ¨ ~60 individuals of known genotype status ¨ within “nuclear” families genetic marker screening n n even distribution/coverage across genome ¨ panel of 263 markers prioritize chromosomes for analysis ¨ comparative biology/genomics

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

comparative genomics

mutation screening n complete DNA sequencing of causative gene ¨ ~140, 000 base pairs ¨ resequencing of animals of known genotype n n 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 n significant portion (30%) of gene absent in affected calves

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.

validation n blind testing of 45 animals of known status ¨ 100% n random testing of ~300 phenotypically normal individuals ¨ none n accurate 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? n Pulmonary Hypoplasia with Anasarca (PHA) ¨ all affected calves from inconsistent sire genotype as homozygotes for identified mutation ¨ all affected calves parentally verify to sire n n except for DNA markers adjacent to causative gene 2 nd mutation – complete deletion of gene ¨ complete deletion of 4 genes (460, 000 bp) ¨ very rare frequency as compared to first

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

pulmonary hypoplasia with anasarca (PHA) n pulmonary hypoplasia n n anasarca n n n tremendous fluid accumulation in affected calves lack of lymphatic development ¨ absence of lymph duct and nodes, athymia invariably lethal n n absent or near absence of lungs ¨ normal cardiovascular system all near term calves born dead other n 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 n recessive Mendelian inheritance n affected pedigrees in both Shorthorn and Maine Anjou breeds

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

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

validation n “blind” testing of 144 animals of known status ¨ 100% n random testing of ~1000 phenotypically normal individuals ¨ none n accurate homozygous for mutation 4 suspect sires test normal ¨ insufficient evidence of their status

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

pedigree assessment n at what point in a pedigree doesn’t it matter anymore? ¨ how n many generations? n (1/2) – 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 n education is key ¨ understand n the possibilities – desired outcome do nothing vs. “kill ‘em all” ¨ up to individual breeders vs. mandatory testing and culling of all carrier animals n accurate identification of carriers ¨ selective vs. comprehensive testing programs ¨ voluntary vs. mandatory

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

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