Inversions in the factor VIII gene in Spanish hemophilia A patients.

Hemophilia A (HA) is an heterogeneous condition both in its clinical severity and at the molecular level. Most of the patients with moderate or mild disease have missense mutations in the factor VI11 gene. In approximately 50% of the severe patients, nonsense or frameshift mutations have been detected. ',* In the remaining severe cases, a defect in the splicing of exon 22 to exon 23 has been demonstrated. ' Intron 22 is the largest intron of the factor VIII gene and contains a CpG island from which two additional transcripts originate. One of these transcripts corresponds to the F8A gene, which has two close telomeric extragenic copies in the X chromosome. An inversion resulting from homologous recombination between the intragenic and the extragenic copies of the F8A has recently been described as a common cause of severe HA.4. 5 We analyzed intron 22 rearrangements in 180 HA patients (1 12 familial and 68 sporadic cases). According to the factor VI11 levels, our sample was classified as severe in 102 cases, as moderate in 27 cases, and as mild in 51 cases. A 0.9-kb EcoWSst I fragment of the intron 22 F8A probe was hybridized to a Southern blot of Bcl I-digested DNA obtained from peripheral blood leukocytes. The normal pattern shows a 21.5-kb band (intragenic gene), a 16-kb band (distal extragenic gene), and a 14-kb band (proximal extragenic gene). Homologous recombination involving the intragenic F8A gene and the distal F8A extragenic copy shows two abnormal bands (20 and 17.5 kb) and the nonmodified 14-kb fragment, whereas recombination with the proximal copy of the gene shows the normal 16-kb band and two modified 20and 15.5-kb fragments5 Our results show (1) an abnormal pattern identifying an inversion in 23% of the analyzed cases (42 of 180). This figure is similar to that of HA patients showing an mRNA abnormality described by Naylor et al? and confirms the hypothesis that at least 20% of HA cases are caused by large inversions in the factor VIII gene. ' (2) Forty-one percent of severe HA patients (42 of 102) showed an inversion. Lakich et al : when describing the inversions, reported this type of mutation in 9 of 19 (47%) severe HA patients. As expected, no inversion was found in our sample of moderate and mild patients. (3) We found a high proportion (31 of 42 [74%]) of the distal rearrangement, as reported by Lakich et al.' (4) From 42 identified inversions, 27 were found in familial cases (27 of 11 2 [24%]), whereas the remaining 15 were detected in sporadic patients (15 of 68 [22%]). This result does not therefore support the suggestion that the inversion is likely to occur de novo in many patients. ' Twenty-three familial cases (85%) showed a distal pattern of rearrangement, whereas only 8 esporadic cases (53%) showed this pattern (Fisher's exact test, P < .03). This high proportion of distal inversions and its preference to occur more frequently in familial than in sporadic cases suggest the existence of a possible etiologic difference between both types of inversions. The distal gene may be more unstable because of its more telomeric localization or because of the presence of nearby recombinogenic sequences. Estimation of the rate at which the proximal or distal rearrangements occur can be obtained by multiplying the estimated mutation rate of the HA (approximately 4 X 10-5)6 by the proportion of hemophilia A patients with the rearrangements (17% for the distal and 6% for the proximal). The figures obtained are about 6.8 X per gene per gamete per generation for the distal rearrangement and 2.4 X for the proximal inversion. (5 ) Inhibitor development was detected in 12 of the cases with an inversion. Goodeve et al' found lack of inhibitor in all 10 patients showing an inversion of the intron 22. Our result suggests that inhibitor development may not be related with the presence or absence of the inversion. From the diagnosis point of view, the identification of such a frequent molecular event in severe HA patients can be applied to carrier and prenatal diagnosis. Because carriers will have the n o d and the altered pattern of bands, a simple Southern blot analysis will considerably improve the provision of accurate genetic counselling in families with the disease.