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.

ABELLEYRO, MM, *PRIMERA AUTORÍA COMPARTIDA; RADIC CP, *PRIMERA AUTORÍA COMPARTIDA; MARCHIONE VD; WAISMAN K; TETZLAFF T; NEME D; ROSSETTI CL; DE BRASI, CD

HUMAN MUTATION

WILEY-LISS, DIV JOHN WILEY & SONS INC

Año: 2020

1059-7794

Hemophilia A (HA) provides excellent models to analyze genotype-phenotype relationships and mutational mechanisms.Aim: Full characterization of 11 non-recurrent/non-homologous F8-large deletions (nhF8ld), performing a comprehensive bioinformatic analysis of DNA elements/features. NhF8ld?s breakpoints were characterized using case-specific DNA-tags, direct- or inverse-PCR amplification and Sanger sequencing. DNA-break?s stimulators (n=46), interspersed repeats, non-B-DNA and secondary-structures were analyzed around breakpoints vs nullhypotheses (E-values) based on computer simulations and base-frequency probabilities. Nine of 18 severe-HA patients (50%) with nhF8lds developed inhibitors, 1/8 affecting one exon and 8/10 (80%) affecting multi-exons. NhF8lds range 2-165-kb. Five nhF8lds (45%)involve F8-extragenic regions including three affecting vicinal genes (SMIM9 and BRCC3) but none shows an extra-phenotype no 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 nhF8lds (27%) show complexities at the breakpoints: an 8-bp inverted-insertion, and the remnant two, inverted- and direct-insertions (46-68-bp) supporting replicative models MMBIR/FoSTeS. The remnant eight nhF8lds (73%) may support NHEJ/MMEJ models.Our study suggests the involvement of the retroposition machinery (e.g., Jurka-targets, Aluelements, LINEs, LTRs), micro-homologies and secondary structures at breakpoints playing significant roles in the origin of the upmost severe phenotype in HA.