Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.
BACKGROUND
Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA.


OBJECTIVE
To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease.


METHODS
Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion.


CONCLUSIONS
The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.
Porada
C D
CD
Department of Animal Biotechnology, University of Nevada, Reno, NV 89557-0104, USA.
Sanada
C
C
Long
C R
CR
Wood
J A
JA
Desai
J
J
Frederick
N
N
Millsap
L
L
Bormann
C
C
Menges
S L
SL
Hanna
C
C
Flores-Foxworth
G
G
Shin
T
T
Westhusin
M E
ME
Liu
W
W
Glimp
H
H
Zanjani
E D
ED
Lozier
J N
JN
Pliska
V
V
Stranzinger
G
G
Joerg
H
H
Kraemer
D C
DC
Almeida-Porada
G
G
D000367:Age Factors; D000375:Aging; D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects / Q000235:genetics*; D003029:Coagulants / Q000494:pharmacology; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D005091:Exons; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D020022:Genetic Predisposition to Disease; D006395:Hemarthrosis / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D006467:Hemophilia A / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010314:Partial Thromboplastin Time; D010641:Phenotype; D012333:RNA, Messenger / Q000097:blood; D027724:Reproductive Techniques, Assisted; D012756:Sheep; D013045:Species Specificity
D003029:Coagulants; D018389:Codon, Nonsense; D012333:RNA, Messenger; C078147:F8 protein, human; D005169:Factor VIII