Molecular insights into the mechanism of non‐recurrent F8 structural variants: full breakpoint characterization and bioinformatics of DNA elements implicated in the upmost severe phenotype in Hemophilia A.

Hemophilia A (HA) provides excellent models to analyze genotype-phenotype relationships and mutational mechanisms. NhF8ld's breakpoints were characterized using case-specific DNA-tags, direct- or inverse-polymerase chain reaction amplification, and Sanger sequencing. DNA-break's stimulators (n = 46), interspersed repeats, non-B-DNA, and secondary structures were analyzed around breakpoints versus null hypotheses (E-values) based on computer simulations and base-frequency probabilities. Nine of 18 (50%) severe-HA patients with nhF8lds developed inhibitors, 1/8 affecting one exon and 8/10 (80%) affecting multi-exons. NhF8lds range: 2-165 kb. Five (45%) nhF8lds involve F8-extragenic regions including three affecting vicinal genes (SMIM9 and BRCC3) but none shows an extra-phenotype not related to severe-HA. The contingency analysis of recombinogenic motifs at nhF8ld breakpoints indicated a significant involvement of several DNA-break stimulator elements. Most nhF8ld's breakpoint junctions showed microhomologies (1-7 bp). Three (27%) nhF8lds show complexities at the breakpoints: an 8-bp inverted-insertion, and the remnant two, inverted- and direct-insertions (46-68 bp) supporting replicative models microhomology-mediated break-induced replication/Fork Stalling and Template Switching. The remnant eight (73%) nhF8lds may support nonhomologous end joining/microhomology-mediated end joining models. Our study suggests the involvement of the retroposition machinery (e.g., Jurka-targets, Alu-elements, long interspersed nuclear elements, long terminal repeats), microhomologies, and secondary structures at breakpoints playing significant roles in the origin of the upmost severe phenotype in HA.