Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 10

Gross DNA Rearrangements

Common Partial Inversion of Factor VIII

The efforts to characterize all mutations in the factor VIII gene in a defined sample of hemophilia A patients revealed a surprising and unexpected finding. After scanning all the exons of factor VIII gene using denaturing gradient gel electrophoresis, Higuchi et al. MLID90152691  72 found the causative mutation in about 90% of patients with mild-to-moderate hemophilia A. 173 However, when severely affected patients were similarly studied, the causative mutation was only found in about 50%. MLID91334474  174 The cause of the remaining 50% remained elusive. Subsequently Naylor et al., MLID93023378  175 using RT-PCR of illegitimate transcription of the factor VIII gene, found that in about 40% of severely affected patients no RT-PCR amplification was possible between exons 22 and 23 of the gene. It was recently demonstrated that these patients have an intrachromosomal inversion due to homologous recombination between the F8A gene in intron 22 and one of two identical copies of F8A located about 550 kb 5' of the factor VIII gene 176 (Fig. 105-5). The elucidation of this hot spot and the development of a simple diagnostic test is of considerable clinical significance since this mutation mechanism accounts for about 25% of all patients with hemophilia A and certainly >40% of those with severe disease.

Large Deletions

In about 5% of the patients with hemophilia A there are large (>50 nucleotides) deletions in the factor VIII gene. MLID89223054  177 The mutation data base of Tuddenham et al. 178 contains 59 different deletions. All deletions characterized have different breakpoints, and there are no two unrelated patients with the same breakpoints, suggesting that the factor VIII gene does not contain sequences that are prone to become deletion breakpoints. Deletions almost always produce severe hemophilia A with no factor VIII activity. A deletion of exon 22 is, however, associated with moderate disease, probably because of in-frame splicing of exons 21 and 23 and production of a protein without the 52 amino acids encoded by exon 22. MLID87231899  179 Few deletion breakpoints have been characterized at the nucleotide sequence level. Most of the breakpoints examined do not occur in repetitive elements such as Alu-sequences. There is usually 2–4 nucleotide homology at the junction point, and the deletion mechanism is probably via nonhomologous recombination. MLID91257856  180

Insertion of Retrotransposons

De novo insertion of LINE repetitive elements in the human genome was first reported in the factor VIII gene. 181 In one case of severe hemophilia A, a 3.8-kb portion of a LINE element was inserted in exon 14 of factor VIII. The inserted DNA had a poly(A)tail, produced a target site duplication, and was inserted in a relatively adenine-rich sequence of exon 14 (Fig. 105-6). The de novo insertion in the second case was a 2.1-kb portion of a LINE element and produced a severe hemophilia A. It occurred in a different site of exon 14 and had all the characteristics of retrotransposition. LINE elements comprise about 5% of the human genome, and there are approximately 105 copies. MLID88050954  182 The full length of the element is 6.5 kb, and most of the copies in the human genome are partial and defective. The consensus sequence of LINE element contains two open reading frames, the second of which predicts a protein with amino acid homology with reverse transcriptase. About 3,000 LINE copies are full length and are potential transposable elements. Only a few of these, perhaps those with open reading frames, can produce a new insertion through an RNA intermediate. They are probably transcribed into DNA and then reinserted as double-stranded DNA into a new genomic site. MLID93253021  183 The full-length "active" LINE element responsible for the insertion in the first hemophilia A patient was cloned and characterized. It maps on chromosome 22 and probably encodes for a peptide that has reverse transcriptase activity. MLID92108429  184 A third LINE element insertion in intron 10 has also been observed but, since it did not co-segregate with the hemophilia A phenotype, it represents a recently established private polymorphism. MLID89233117  185 Insertions of LINE or other retrotransposons are not common, since there are only two such examples in >1,000 patients studied.