Structural variants in the human genome: lessons from hemophilia. Analysis of the SV mechanisms by characterization of breakpoints and bioinformatics of the involved sequences.

Hemophilia A (HA) and B (B) are recessive X-linked coagulopathies, whose maximum clinical severity is caused, in a large percentage of cases, by structural variants (SV) associated with HA, such as the intron 22 inversion (Inv22), of the intron 1 (Inv1) (40-50%) or large F8 deletions (8-15%), and with HB, such as large F9 deletions (3-10%). Specific amplification of SV breakpoints allows detailed characterization of neighboring sequences potentially involved in the original event. Among SVs, large deletions of F8 and F9 can be detected by the consistent absence of specific amplification signals in hemizygous males. This primary identification in hemizygosity allows addressing the delimitation of the 5' and 3' breaks using localized amplification STS and the characterization of the junctions by gap-PCR amplification and Sanger sequencing. These cases contribute to a very scarce collection of genomic rearrangements characterized and analyzed in the literature. The localization of the breakpoints on the reference genome, permits identifying recombination-stimulating motifs (generators of single and/or double-strand breaks) and the bioinformatic/statistical analysis, including null hypotheses to evaluate their probability, ultimately estimating the mechanisms involved in each SV. Among the mechanisms that have been associated with the origin of SV in humans, and hemophilia causative SVs reported by us and others, are those associated with DNA replication, such as Micro-homology mediated break induced repair (MMBIR) and fork stalling and template switching. (FoSTeS) and those associated with meiosis, such as non-homologous end joining (NHEJ) and non-allelic homologous recombination (NAHR) mediated by duplicons. Likewise, this topic includes the Inv22 analysis, its mechanism of origin, its complex HGVS nomenclature and the genotyping of all its types (1 and 2) and variants (1v and 2v) and all rearrangements mediated by int22h duplicons, such as the duplications (Dup22) and deletions (Del22), which encompasses a 0.5 Mb region on Xq28 with more than six medically important genes in addition to F8.