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.

中文翻译：

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非周期性F8结构变异机制的分子见解：涉及A型血友病最严重表型的DNA元件的完整断点表征和生物信息学

血友病A（HA）提供了出色的模型来分析基因型与表型的关系和突变机制。Nh F8 ld的断点通过案例特异的DNA标签，直接或反向聚合酶链反应扩增和Sanger测序来表征。 基于计算机模拟和基频概率，分析了断裂点与无效假设（E值）周围的DNA断裂刺激物（n = 46），散布的重复序列，非B DNA和二级结构。nh F8 lds的18例重度HA患者中有9例（50％）产生抑制剂，其中1/8影响一个外显子，8/10（80％）影响多外显子。Nh F8 lds范围：2-165 kb。五个（45％）Nh F8 lds涉及F8包括三个受影响的邻近基因（SMIM9和BRCC3）的外源区域，但没有一个区域显示出与严重HA不相关的表型。对在nh F8 ld断裂点的重组基序进行的权变分析表明，一些DNA断裂刺激因子大量参与。大多数nh F8 ld的断点连接均表现出微同源性（1–7 bp）。三个（27％）nh F8 lds在断点处表现出复杂性：一个8 bp的反向插入，以及剩余的两个，反向和直接插入（46-68 bp）支持复制模型的微同源性介导的断裂诱导的复制/货叉停转和模板切换。剩余八（73％）Nh F8lds可能支持非同源末端连接/微同源介导的末端连接模型。我们的研究表明，逆转机制（例如，Jurka靶标，Alu元素，较长的散布的核元素，较长的末端重复序列），微观同源性和断裂的二级结构的参与在HA最严重的表型起源中起着重要作用。