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.
Haemophilia A (HA) (OMIM #306700), an X-linked recessive disorder characterized by reduced activity of coagulation factor VIII (FVIII:C), is caused by deleterious mutations in the F8. HA can be treated by administration of the deficient FVIII. However, about 20%-30% of severe HA patients (biochemically defined as FVIII:C < 1 IU/dL) developed FVIII neutralizing antibodies (inhibitors) making replacement therapy ineffective. Inhibitors result in higher therapy costs and decreased quality-of-life and life expectancy of patients with haemophilia. From the Public Health System perspective, Argentina currently compiles 96 HA patients with clinically identified inhibitors out of a total 2220 [1]. This reduced figure (96/2220) is a successful result of undergoing an extended prophylaxis covering more than 65% of patients, in which therapeutic FVIII is not administered under risk conditions, and immune-tolerance induction treatment for inhibitor eradication.As a typical complex trait (multifactorial) an operative classification of inhibitor development in haemophilia focuses on two main groups of risk factors: modifiable (environmental factors) and non-modifiable (genetic factors, often involving several genes with different relative weight predisposing to the phenotype). Among the former, environmental risk factors include treatment-related factors and immune-system challenges. Among the latter, in HA, the causative F8 genotype has been established as the main factor conditioning inhibitor development (in Argentina [2], and worldwide, reviewed in [3]), but it also counts a group of secondary risk factors, weaker than the F8 genotype, such as family history of inhibitors, ethnicity, human lymphocyte antigen haplotype and polymorphisms linked to immune-system genes, such as
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