The Chapel Hill hemophilia A dog colony exhibits a factor VIII gene inversion - PubMed (original) (raw)

The Chapel Hill hemophilia A dog colony exhibits a factor VIII gene inversion

Jay N Lozier et al. Proc Natl Acad Sci U S A. 2002.

Abstract

In the Chapel Hill colony of factor VIII-deficient dogs, abnormal sequence (ch8, for canine hemophilia 8, GenBank no. ) follows exons 1-22 in the factor VIII transcript in place of exons 23-26. The canine hemophilia 8 locus (ch8) sequence was found in a 140-kb normal dog genomic DNA bacterial artificial chromosome (BAC) clone that was completely outside the factor VIII gene, but not in BAC clones containing the factor VIII gene. The BAC clone that contained ch8 also contained a homologue of F8A (factor 8 associated) sequence, which participates in a common inversion that causes severe hemophilia A in humans. Fluorescence in situ hybridization analysis indicated that exons 1-26 normally proceed sequentially from telomere to centromere at Xq28, and ch8 is telomeric to the factor VIII gene. The appearance of an "upstream" genomic sequence element (ch8) at the end of the aberrant factor VIII transcript suggested that an inversion of genomic DNA replaced factor VIII exons 22-26 with ch8. The F8A sequence appeared also in overlapping normal BAC clones containing factor VIII sequence. We hypothesized that homologous recombination between copies of canine F8A inside and outside the factor VIII gene had occurred, as in human hemophilia A. High-resolution fluorescent in situ hybridization on hemophilia A dog DNA revealed a pattern consistent with this inversion mechanism. We also identified a HindIII restriction fragment length polymorphism of F8A fragments that distinguished hemophilia A, carrier, and normal dogs' DNA. The Chapel Hill hemophilia A dog colony therefore replicates the factor VIII gene inversion commonly seen in humans with severe hemophilia A.

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Figures

Figure 1

Chapel Hill canine factor VIII exon 22/ch8 junction. In the Chapel Hill hemophilia A canine factor VIII transcript, the novel sequence ch8 follows exon 22. The predicted amino acid sequence is shown in three-letter code below the nucleic acid sequence data. The ORF of exon 22 continues 22 amino acids after canine factor VIII Met 2116 before the first of several stop codons (depicted as periods) in ch8. Sites of cleavage by various restriction sites used to characterize the genomic DNA or generate DNA probes are shown above the DNA sequence. pA, the polyadenylation signal sequence, AATAAA.

Figure 2

Metaphase FISH Analysis of hemophilia A and normal dogs with ch8 and factor VIII BAC clone probes. FISH of normal and hemophilia A dog chromosomes with BAC clones specific for canine factor VIII exons 1–22 (292 C4) or the novel ch8 sequence (291 M9) localizes both sequences to Xq28. 292 C4 (containing canine factor VIII exons 1–22) is red and 291 M9 (containing the novel ch8 sequence) is green.

Figure 3

DNA fiber FISH analysis of hemophilia A and normal dogs with ch8 and factor VIII BAC clone probes. FISH of normal and hemophilia A dog genomic DNA with factor VIII and related BAC probes is shown. BAC clone 291 M9 (which is outside of the factor VIII gene and contains both ch8 (the 3′ end of the abnormal hemophilia A factor VIII transcript) and F8A (the factor VIII-associated gene sequence) is green. BAC clone 292 C4 (which contains factor VIII exons 1–22 as well as a copy of F8A) is blue. BAC clone 314 016, which contains factor VIII exons 23–26 and one copy of F8A, is red. BAC clones 292 C4 and 314 O16 presumably overlap in the vicinity of intron 22. BAC clones 292 C4 and 314 O16 establish the orientation of factor VIII exons 1–22 and 23–26. Rearrangement of DNA is evident from inversion of part of the 291 M9 probe with part of that for 292 C4 as well as the shift in the normal gap between extragenic probe 291 M9 and factor VIII probes 292 C4 and 314 O16.

Figure 4

A _Hin_dIII RFLP of F8A fragments distinguishes hemophilia A, carrier female, and normal dog genomic DNA. The 28- and 13-kb _Hin_dIII bands found in normal dog genomic DNA (N) are not found in hemophilia A dog DNA (H), which demonstrates bands at 21 and 23 kb. The carrier female (C) has all four bands seen in hemophilia A or normal dog DNA. The normal dog BAC clone 291 M9 has the 28-kb band, suggesting the 28-kb fragment is extragenic to factor VIII. Normal dog BAC clones 292 C4 and 314 O16 contain the 13-kb band, suggesting the 13-kb fragment is within factor VIII, presumably in intron 22 where these BACs overlap. Not shown are bands at ≈5 and ≈3.5 kb that are present in all three genomic DNA samples (see Fig. 11).

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The Chapel Hill hemophilia A dog colony exhibits a factor VIII gene inversion - PubMed (original) (raw)