Localization of Non-Factor VIII Sequence Involved in the Factor VIII Gene Inversion in Hemophilia A Dogs.

There is a common gene inversion at the telomere of the canine X chromosome in which factor VIII intron 22 DNA recombines with homologous sequence outside the gene. This mutation disrupts factor VIII synthesis and causes severe hemophilia A in dogs, analogous to a common inversion seen in humans (Lozier et al, PNAS 2002 99:12991–6). This mutation appears to be the only spontaneous gene inversion in an animal that replicates a corresponding disease of humans. The coding region for canine factor VIII spans approximately 146 kb and is found at the telomere of the X chromosome between bases 126,063,525 and 125,917,394 in the canine genome project, version 46.2d. Much of the canine factor VIII intron 22 remains unsequenced and the presence of F8A (the putative site of recombination in the factor VIII gene) in this region is inferred from PCR and Southern blot analysis of BAC clones that encompass this region. We have therefore focused on localizing the non-factor VIII site of the recombination and have found its essential elements in sequence located telomeric (upstream) to the canine factor VIII coding start site. We have used the boxer canine genome sequence (version 46.2d) as a template and reference for sequence analysis of BAC clone 291M9 (from normal Doberman pinscher genomic DNA library RPCI-81). Comparison of >100,000 bp of sequence common to the boxer and Doberman in this region showed >99% identity between the two breeds as expected. The BAC clone 291M9 was previously shown by DNA fiber FISH to be the region outside the factor VIII gene that participates in the gene inversion. This clone contains both F8A sequence (the putative site of recombination) as well as the sequence (ch8) that replaces the last four exons of the factor VIII transcript in hemophilia A dogs after the inversion. F8A sequence found in the Doberman BAC clone 291M9 matches sequence starting at nucleotide 126,317,916, in the boxer canine genome project (from RPCI-82 boxer BAC clone XX-145E20). This is outside the factor VIII gene, and approximately 400,000,000 base pairs upstream/telomeric to F8A found within the factor VIII gene. Approximately 20 kb from F8A is ch8, the sequence spliced into the abnormal hemophilia A factor VIII mRNA. The 7.5 kb region where the F8A sequence is found is extremely rich in guanosine and cytosine nucleotides (73% GC content) and has at least six putative CpG islands (as identified by the EMBOSS CpG analysis software (http://www.ebi.ac.uk/emboss/cpgplot/). This is consistent with the finding that F8A gene sequences outside the human factor VIII gene are found in CpG islands. There may be additional copies of the canine F8A sequence in or near this region (as is the case for the human genome), since gaps remain in this region of the known genomic sequence. The high GC content and highly repetitive nature makes sequence analysis and assembly problematic. These properties are consistent with DNA that is susceptible to homologous recombination and gene inversion as is seen with hemophilia in dogs and man.

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