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Molecular insights into the mechanism of nonrecurrent F8 structural variants: Full breakpoint characterization and bioinformatics of DNA elements implicated in the upmost severe phenotype in hemophilia A
Author(s) -
Abelleyro Miguel Martín,
Radic Claudia Pamela,
Marchione Vanina Daniela,
Waisman Karen,
Tetzlaff Tomas,
Neme Daniela,
Rossetti Liliana Carmen,
De Brasi Carlos Daniel
Publication year - 2020
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.23977
Subject(s) - breakpoint , biology , genetics , sanger sequencing , inverted repeat , dna , phenotype , exon , gene , dna sequencing , computational biology , microbiology and biotechnology , genome , chromosome
Hemophilia A (HA) provides excellent models to analyze genotype–phenotype relationships and mutational mechanisms. Nh F8 ld'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 nh F8 lds developed inhibitors, 1/8 affecting one exon and 8/10 (80%) affecting multi‐exons. Nh F8 lds range: 2–165 kb. Five (45%) nh F8 lds 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 nh F8 ld breakpoints indicated a significant involvement of several DNA‐break stimulator elements. Most nh F8 ld's breakpoint junctions showed microhomologies (1–7 bp). Three (27%) nh F8 lds 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%) nh F8 lds 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.