A Frame Shift-Induced Stop Codon Causes Hemophilia a in Sheep.

Hemophilia A (HA), or Factor VIII (FVIII) deficiency, is the most common severe hereditary coagulation disorder, affecting 1 in 5000 males. We have successfully re-established and characterized a line of sheep with a bleeding disorder that closely mimics severe human HA. These animals have almost non-existent levels of FVIIIc and an extremely prolonged PTT, with normal levels of platelets, fibrinogne, FVII, FIX, and vWF. All animals that survived birth have exhibited prolonged tail and nail cuticle bleeding, multiple episodes of severe spontaneous bleeding including hemarthroses, muscle hematomas, and hematuria, all of which have responded to recombinant human FVIII. Thus far, low-titer inhibitors to human FVIII have been detected in 3 treated animals, further validating the clinical relevance of this model. Unfortunately, almost nothing is known about the sheep FVIII gene or about the nature of the mutation causing HA in these animals. We thus undertook to characterize the normal sheep FVIII mRNA and elucidate the nature of the hemophilia-inducing mutation. RT-PCR of mRNA from the spleen of normal sheep, followed by overlapping sequence analysis allowed us to walk along the mRNA and obtain the sequence of the complete coding sequence for factor VIII. The coding sequence for ovine FVIII is 6765 nucleotides, which is translated into a protein consisting of 2254 amino acids (a.a.). BLAST alignment of this sequence to that of human FVIII at the a.a. level revealed a high degree of identity in all regions except the B domain (which in humans is known to be dispensable for clotting activity). Specifically, the A1 domain (ovine a.a. 1–332) showed 81% identity, A2 (a.a. 333–709) 88%, A3 (a.a. 1596–1924) 87%, C1 (a.a. 1925–2074) 90%, and C2 (a.a. 2075–2254) 86%, while the B domain (a.a. 710–1595) exhibited only 47% identity. A comparison of these sequences taking into consideration conservative a.a. changes that maintain the chemical properties of sheep and human FVIII proteins indicated even higher levels of homology, with the A1 domain exhibiting 87% positivity, A2 94%, A3 93%, C1 94%, and C2 93%. Even with this approach, however, there was still very low conservation (59%) of the B domain between the two species. Analysis of mRNA isolated from the spleen of a deceased HA sheep revealed several conservative point mutations in the hemophiliac and identified 11bp in exon 14 that differed between the wild type and the hemophiliac. Importantly, this difference included a frame-shift that introduced a premature stop codon in exon 14, as is seen in some human patients with HA. Using a PCR-based RFLP analysis, we can unequivocally distinguish sheep that are wild-type, heterozygous, or homozygous for the HA mutation, and thus confirm the genotype of all 26 existing carriers and 8 hemophiliac sheep. This PCR-based assay will greatly facilitate studies using these sheep, since it is now possible to either screen embryos prior to implantation or screen fetuses in utero using a small volume of blood or amniotic fluid, as is done for prenatal diagnosis of human genetic diseases. These studies thus defined the molecular basis for the HA in these sheep that are a valuable model of human disease. Given the close physiologic similarity between sheep and humans, the high degree of identity in their FVIII protein, and the decades of experience using the sheep to study both normal physiology and a wide array of diseases, we hope that this large animal model will contribute to a better understanding of HA and the development of novel treatments such as stem cell transplantation and gene therapy-based approaches that can directly translate to human patients with hemophilia.