Aberrant F8 gene intron 1 inversion with concomitant duplication and deletion in a severe hemophilia A patient from Southern Italy

Abstract

Intron 1 and intron 22 inversions (inv1 and inv22) represent the most prevalent mutations in the F8 gene (F8) (1-5% and 40-45%, respectively) causative of severe haemophilia A (HA) [1]. These alterations result from intrachromosomal recombination between intronic regions (int1h-1 and int22h-1, respectively) and their homologous extragenic copies in the telomeric position (int1h-2 and int22h-2/3, respectively) [1]. Inv1 causes an altered F8 structure with the translocation and inversion of the 5¢ region including exon 1 in the extragenic site, giving rise to two chimeric mRNAs: one containing the promoter and first exon of the F8 followed by some exons of the VBP1 gene; the other one formed by the promoter and coding sequences of the C6.1A gene joined to a part of the intron 1 and exons 2-26 of the F8 [2]. The clinical effect of this rearrangement is a severe phenotype with the absence of functional factor (F)VIII. According to literature data, the prevalence of inv1 ranges from 0% to 5% in severe HA patients from different countries [3,4]. In our cohort of 135 unrelated severe HA patients from Southern Italy, inv1 was found in four patients (2.9%) [5]. Here, we report the case of a severe HA patient, who showed an unusual pattern for inv1 associated with a complex gene rearrangement not described to date.The propositus is a Caucasian young male diagnosed with severe HA at the age of 1 year because of a positive family history. The causative F8 mutation in his affected uncle or the carrier status in his mother had not been previously searched for. The patient was treated with recombinant FVIII concentrates with no personal or family history of inhibitor development. We first analyzed the patient for both F8 inversions. This analysis excluded inv22 but showed a novel abnormal pattern for inv1, compared with those previously reported in literature, with two bands: one corresponding to the normal int1h-1 region (1908 bp) and the other corresponding to the band with the inversion of the int1h-1 sequence (1323 bp), as a heterozygous female, suggesting the presence of more than two copies of the int1h region (Fig. 1A). No bands of the int1h-2 region were detected, indicating a deletion within the homologous extragenic copy or a new rearrangement of sequences flanking the int1h-2 repeat not detectable by the PCR method used (Fig. 1A). We also investigated the presence of inv1 in his relatives: both mother and sister were carriers of the same aberrant pattern plus the 1191-bp fragment specific for the normal homologous extragenic int1h-2 sequence. The affected uncle was not available for molecular analysis. To confirm the direct diagnosis, linkage analysis was performed in this family, using intragenic (intron 18 BclI and intron 19 HindIII RFLP) and extragenic (DXS52 VNTR) polymorphisms, matching up with the same genetic profile. Furthermore, in the patient sequencing analysis of the F8, including all 26 exons, flanking intron/exon boundaries, promoter, 5¢ and 3¢ regions, did not reveal other mutations. In this family, a...

Tandem inversion duplication within F8 Intron 1 associated with mild haemophilia A

“…To date, no study has reported this large duplication of the F8 exon 1 in the international HA mutation database [http://www.eahad-db.org/], in the scientific literature, or in the CNV database [http://projects.tcag.ca/variation/], nor has it been described in studies of over 300 patients from the general Belgian population. Nevertheless, three complex rearrangements involving severe HA patients have previously been reported describing duplications of F8 intron 1 that, contrary to our cases, were associated with a classical inversion of intron 1 . Recessive von Willebrand disease type 2 Normandy, transmitted as recessive disorder, could theoretically be ruled out, given the clearly inherited X‐linked recessive HA in the three families that has been present for at least four generations.…”

Molecular cytogenetic characterization of five F8 complex rearrangements: utility for haemophilia A genetic counselling

“…Genomic inversion does not normally result in gain or loss of DNA, but unusual patterns have been described in Inv22 and Inv1 that lead to the hypothesis of more complex rearrangements involving deletions or duplications [14][15][16][17][18][19][20][21][22][23][24][25][26]. Furthermore, mechanisms causing these complex rearrangements are poorly understood.…”

Review of molecular mechanisms at distal Xq28 leading to balanced or unbalanced genomic rearrangements and their phenotypic impacts on hemophilia

“…Others are mediated by int22h/int1h copies without producing inversions . These have all been identified during F8 routine genetic analyses of Inv22 and Inv1 and classified as aberrant Inv patterns . They consist of both deletions and duplications, and could be more common than expected.…”

“…This additional event could be missed by routine genetic analyses as usual methods for Inv detection (southern blot or polymerase chain reaction [PCR] approaches) only detect DNA sequences around inth copies. This is unfortunate, as the combination of int22h ‐related inversion and large deletion in the F8 gene could be associated with X‐linked moyamoya angiopathy syndrome, and could additionally significantly increase the risk for developing high levels of inhibitors . It has been shown that genotypes with Inv22 maintain the possibility of F8 transcription.…”

“…This is unfortunate, as the combination of int22h-related inversion and large deletion in the F8 gene could be associated with X-linked moyamoya angiopathy syndrome, and could additionally significantly increase the risk for developing high levels of inhibitors. 11,[46][47][48] It has been shown that genotypes with Inv22 maintain the possibility of F8 transcription. Indeed, this transcription results in a polyadenylated fusion transcript containing intact FVIII exons 1-22 plus 16 additional non-FVIII amino acids at its C-terminal end.…”